Cannabinoid receptor 2 (CB2) inverse agonists and therapeutic potential for multiple myeloma and osteoporosis bone diseases

ABSTRACT

Cannabionid receptor-2 inverse antagonists include compounds represented by Formula IV, or a pharmaceutically acceptable salt thereof: 
     
       
         
         
             
             
         
       
         
         
           
             wherein: R 1  and R 2  are independently H, alkyl, or alkenyl; R 3  is alkyl, alkenyl, aryl, aralkyl, aralkenyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl; R 4  and R 5  are independently a bond, alkylenyl, or alkenylenyl; each R 6  and R 7  is independently selected from the group consisting of OH, F, Cl, Br, I, (C 1 -C 6 )alkyl, alkoxy, amino, COOH, CONH 2 , SO 3 H, PO 3 H 2 , CN, SH, NO 2  and CF 3 ; and p and q are independently 0, 1, 2, 3, 4, or 5. Such compounds may be used to treat osteoporosis or multiple myeloma.

This application is a divisional of U.S. patent application Ser. No.13/715,603, filed on Dec. 14, 2012, now U.S. Pat. No. 8,772,541, whichclaims priority from U.S. Provisional Patent Application No. 61/576,041,filed on Dec. 15, 2011, all of which are incorporated herein byreference in their entirety.

FIELD

The present invention relates to compounds by the regulation ofcannabinoid receptors. More specifically, the compounds are inverseantagonists of cannabinoid receptor subtype-2, otherwise known as CB2.

BACKGROUND

Osteoporosis is a major metabolic bone disease that affects 44 millionAmericans or 55% of the people 50 years of age or older, among which 10million individuals already have this disease and the rest 34 millionmore at increased high risk for osteoporosis. The disease causes asignificant amount of morbidity and mortality in patients and is oftendiagnosed after a fracture occurs.

The endocannabinoid system plays an important role in regulatingskeletal remodeling and bone mass [2, 3]. These physiological processesare implicated to play a role in the development and progression ofosteoporosis. In particular, CB2-deficient mice show a remarkablyaccelerated age-related bone loss, supported by human genetic studiesthat portray polymorphisms in CNR2 gene (encoding CB2) as importantgenetic risk factors for osteoporosis. However, CB2-mediated boneanabolic action as well as the underlying mechanisms has not been fullyexplored. The present application provides compounds that can be used asprobes to study the mechanisms involved in CB2-mediated regulation ofosteoporotic signaling.

Multiple myeloma (MM), an incurable cancer of plasma cells is the secondmost common hematological malignancy in the United States. The diseasedisproportionately affects males over females, and is more common in theAfrican American population than in Caucasians. The etiology of MM isunknown, and at present, there is no cure available, although moderntreatment regimens have been able to slow disease progression in manypatients, and have extended survival rates to about 3-5 yearspost-diagnosis.

MM patients present various symptoms, including hypercalcemia, anemia,renal failure, and impaired production of non-pathologicalimmunoglobulins. Many patients also endure persistent bone pain, whichtypically stems from small fractures in the bones. Indeed, the hallmarkpathology of MM is increased bone destruction and development ofosteolytic lesions, which are mediated by high osteoclast (OCL) activityand make the patient more susceptible to bone fractures.

Previous work from the present inventors revealed that compoundsbelonging to the chemical genus shown below modulated cannabinoidreceptor-2 activity. Preliminary biological data illustrates that thisclass of compounds to selectively modulate the CB2 receptor.

The cannabinoid receptor subtypes CB1 (brain) and CB2 (spleen) areimportant G-protein coupled receptor targets for developing newtherapeutic agents. Since the discovery of the cannabinoid (CB)receptors, their endogenous ligands, and enzymes implicated to play arole in cannabinoid receptor and ligand biology there has been intensivepharmacological research into the therapeutic potential of cannabinergicligands.

Clinical data related to the therapeutic potential of CB ligands for thetreatment of nausea, glaucoma, cancer, stroke, pain, neuronal disorders,osteoporosis, multiple sclerosis, and autoimmune disorders has generatedactive interest in cannabinoid research. While most of the researchefforts have focused on the development of ligands targeted to the CB1receptor, biological data indicates that CB 1 ligands exert undesirablepsychotropic side effects. These side effects have caused publicconcern. However, work to design novel CB2 ligands that do not conferpsychotropic side effects associated with modulation of CB 1 activityhas been limited, largely due to a lack of information about the threedimensional structures of the CB receptors and ligand binding sites.

The present inventors have used structure-activity relationship (SAR),studies to explore and define the chemical space of CB2 receptor that isinvolved in ligand binding interactions. Early studies used to definethis chemical space relied on QSAR/NMR methodologies and in-silicodocking experiments to identify a library of chemically diversescaffolds as the core pharmacophore for CB2 receptor ligand design.

SUMMARY

The present invention uses SAR information to develop a new classcompounds that selectively target and modulate CB2 activity,particularly with an aim to developing small molecule therapeutics fortreating multiple myeloma and osteoporosis. Compounds according to thepresent invention also provide alternate therapeutic candidates tocurrent medications, such as bisphosphonates, raloxifene, calcitonin andhormone replacement therapy that are used to treat osteoporosis and areknown to exhibit severe adverse effects which limits their clinical use.

According to an embodiment of this invention, therefore, is provided acompound according to Formula I or a pharmaceutically acceptable saltthereof.

For Formula I compounds D and D′ are independently —H, —OH, —OR^(a),(C₁-C₆)alkyl or

and R^(a) is H, straight or branched chain (C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C₃-C₁₄)aryl,(C₃-C₁₄)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₁₄)aryl(C₁-C₆)alkylene-.

In Formula I substituent groups A, B and Q are each independently(C₁-C₆)alkylene, (C₂-C₆)alkenylene or (C₂-C₆)alkynylene and subscriptse, f and g independently are integers between 0 and 6 inclusive.

V, W, X, Y, and Z are each independently a bond, —C(R′″)₂—, —CR′″—,—NR′″—, —N—, —O—, —C(O)—, or —S—, with the proviso that no two adjacentmembers of V, W, X, Y, and Z are simultaneously —O—, —S—, or —NR′″—.

In Formula I, R′″ is H, —OH, —OR^(a), halogen, (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, —NH₂, —NH(C₁-C₆)alkyl, —N[(C₁-C₆)alkyl]₂, —CN,(C₃-C₈)heteroaryl, (C₃-C₈)heterocycloalkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl, (C₃-C₈)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₈)heteroaryl-(C₁-C₆)alkylene-, (C₃-C₈)aryl(C₁-C₆)alkylene-,(C₃-C₈)aryl(C₁-C₆)alkenylene-, or (C₁-C₆)alkyl-(C₃-C₈)arylene andsubscripts l, m, n, p and q independently are integers between 0 and 2inclusive, with at least one of l, m, n, or p is not 0.

represents the option of having one or more double bonds. For compoundsthat conform to Formula I, any alkyl, alkylene, alkenylene, aryl,heteroaryl, cycloalkyl, or heterocycloalkyl is optionally substitutedwith halogen, oxo, —COOH, —CN, —NO₂, —OH, —NR^(d)R^(e), (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl, (C₃-C₈)aryl,(C₃-C₈)heteroaryl, (C₃-C₈)heterocycloalkyl, or (C₃-C₈)aryloxy; withR^(d) and R^(e) each independently being H, straight or branched(C₁-C₆)alkyl, optionally substituted (C₃-C₈)aryl, optionally substituted(C₃-C₁₄)aryl(C₁-C₆)alkylene-, and H₂N(C₁-C₆)alkylene-.

When each

in Formula I is independently a phenyl, B_(f) and Q_(g) are bothmethylene and e is 0 then D is not 4-dimethylaminophenyl group.

Exemplary Formula I compounds are those illustrated in the table below:

Another embodiment of the invention is a compound according to FormulaI′ or a pharmaceutically acceptable salt thereof:

In Formula I′, D and D′ are independently —H, —OH, —OR^(a), (C₁-C₆)alkylor

Substituents R^(a′), R^(a″) and R^(a′″) are independently selected fromthe group consisting of H, straight or branched chain (C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C₃-C₁₄)aryl,(C₃-C₁₄)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, and (C₃-C₁₄)aryl(C₁-C₆)alkylene-.

A, B and Q are each independently (C₁-C₆)alkylene, (C₂-C₆)alkenylene or(C₂-C₆)alkynylene.

Subscripts e, f and g independently are integers between 0 and 6inclusive.

Ring members V, W, X, Y, and Z are each independently a bond, —C(R′″)₂—,—CR′″—, —NR′″—, —N—, —O—, —C(O)—, or —S—, wherein no two adjacentmembers of V, W, X, Y, and Z are simultaneously —O—, —S—, or —NR′″—.

In Formula I′, R′″ is H, —OH, —OR^(a), halogen, (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, —NH₂, —NH(C₁-C₆)alkyl, —N[(C₁-C₆)alkyl]₂, —CN,(C₃-C₈)heteroaryl, (C₃-C₈)heterocycloalkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl, (C₃-C₈)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₈)heteroaryl-(C₁-C₆)alkylene-, (C₃-C₈)aryl(C₁-C₆)alkylene-,(C₃-C₈)aryl(C₁-C₆)alkenylene-, or (C₁-C₆)alkyl-(C₃-C₈)arylene.

Subscripts l, m, n, p and q independently are integers between 0 and 2inclusive, wherein at least one of l, m, n, or p is not 0.

Also in Formula I′, the symbol

represents the option of having one or more double bonds.

In Formula I′ compounds, any alkyl, alkylene, alkenylene, aryl,heteroaryl, cycloalkyl, or heterocycloalkyl is optionally substitutedwith halogen, oxo, —COOH, —CN, —NO₂, —OH, —NR^(d)R^(e), (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl, (C₃-C₈)aryl,(C₃-C₈)heteroaryl, (C₃-C₈)heterocycloalkyl, or (C₃-C₈)aryloxy; and R^(d)and R^(e) are each independently H, straight or branched (C₁-C₆)alkyl,optionally substituted (C₃-C₈)aryl, optionally substituted(C₃-C₁₄)aryl(C₁-C₆)alkylene-, and H₂N(C₁-C₆)alkylene-.

According to another embodiment is provided a compound according toFormula II or a pharmaceutically acceptable salt thereof.

According to Formula II, A, B′ and E are each independently a bond,—C(R′″)₂—, —CR′″—, —NR′″—, —N—, —O—, —C(O)—, or —S— and no two adjacentmembers of A, B′ and E are simultaneously —O—, —S—, or —NR′″—.Subscripts h, j and k independently are integers between 0 and 2inclusive, with at least one of h, j or k not being 0.

represents the option of having one or more double bonds and D and D″are each independently —C(O), —CH₂C(O)—, (C₁-C₆)alkylene, —C(O)NH—, or—NHC(O)—.

For Formula II compounds,

represents the option of having a C₅-C₆ fused ring optionally having oneor more double bonds.

R and R₁ are each independently OH, —OR^(a), (C₁-C₆)alkyl,(C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C₈)heteroaryl,(C₃-C₈)heterocycloalkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl,(C₃-C₈)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₈)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₈)aryl(C₁-C₆)alkylene- andR^(a) is H, straight or branched chain (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,(C₃-C₁₄)aryl, (C₃-C₁₄)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₁₄)aryl(C₁-C₆)alkylene-.

For compounds according to Formula II, any alkyl, alkylene, aryl,heteroaryl, cycloalkyl, or heterocycloalkyl is optionally substitutedwith one or more of halogen, oxo, —COOH, —CN, —NO₂, —OH, —NR^(d)R^(e),(C₁-C₆)alkoxy, or (C₃-C₈)aryloxy; with R^(d) and R^(e) eachindependently being H, straight or branched (C₁-C₆)alkyl, optionallysubstituted (C₃-C₁₄)aryl(C₁-C₆)alkylene-, and H₂N(C₁-C₆)alkylene-.

Exemplary Formula II compounds include

The invention also provides in another embodiment a compound accordingto Formula III′ or a pharmaceutically acceptable salt thereof:

In Formula III′, X is N,N⁺, or —CH—.

R′ is H, straight or branched chain (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,(C₃-C₁₄)aryl, (C₃-C₁₄)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₁₄)aryl(C₁-C₆)alkylene-.

Q′, R and T are each independently selected from a bond,—C(O)(C₁-C₆)alkylene-, (C₁-C₆)alkyl, —S(O)₂—, —S(O)—, —S(O)₂NHR″,—O—(C₁-C₆-alkylene)-O—, —OC(O)— and —(C₁-C₆-alkylene)-OC(O)—.

G, H, J, L, and M are each independently a bond, —C(R′″)₂—, —CR′″—,—NR′″—, —N—, —O—, —C(O)—, and—S—, wherein no two adjacent members of G,H, J, L, or M are simultaneously —O—, —S—, or —NR′″—.

R′″ is H, —OH, —OR^(a), halogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,—NH₂, —NH(C₁-C₆)alkyl, —N[(C₁-C₆)alkyl]₂, —CN, (C₃-C₈)heteroaryl,(C₃-C₈)heterocycloalkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl,(C₃-C₈)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₈)heteroaryl-(C₁-C₆)alkylene-, (C₃-C₈)aryl(C₁-C₆)alkylene-, or(C₁-C₆)alkyl-(C₃-C₈)arylene;

Subscripts l, m, n, p and q independently are integers between 0 and 2inclusive, wherein at least one of l, m, n, or p is not 0. Subscript ois 0 when X is N or CH, and o is 1 when X is N⁺.

The symbol

represents the option of having one or more double bonds.

R^(a) is H, straight or branched chain (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,(C₃-C₁₄)aryl, (C₃-C₁₄)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₁₄)aryl(C₁-C₆)alkylene-.

In Formula III′, any alkyl, alkylene, alkenylene, aryl, heteroaryl,cycloalkyl, or heterocycloalkyl is optionally substituted with one ormore of halogen, oxo, —COOH, —CN, —NO₂, —OH, —NR^(d)R^(e), (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl, (C₃-C₈)aryl,(C₃-C₈)heteroaryl, (C₃-C₈)heterocycloalkyl, or (C₃-C₈)aryloxy; R^(d) andR^(e) are each independently H, straight or branched (C₁-C₆)alkyl,optionally substituted (C₃-C₈)aryl, optionally substituted(C₃-C₁₄)aryl(C₁-C₆)alkylene-, or H₂N(C₁-C₆)alkylene-.

The present invention also provides a compound according to Formula IIIor a pharmaceutically acceptable salt thereof

For Formula III compounds X is N, N⁺, or —CH—. R′ is H, straight orbranched chain (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₁₄)aryl,(C₃-C₁₄)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₁₄)aryl(C₁-C₆)alkylene-.

Substituent groups Q′, R and T are each independently (C₁-C₆)alkyl,—S(O)₂—, —S(O)—, —S(O)₂NHR″, —O—(CH₂)_(x)—O—, —OC(O)— and(CH₂)_(x)—OC(O)—, while substituent groups G, H, J, L, and M eachindependently being a bond, —C(R′″)₂—, —CR′″—, —NR′″—, —N—, —O—, —C(O)—,and —S—. For Formula III compounds no two adjacent members of G, H, J,L, or M are simultaneously —O—, —S—, or —NR′″—.

R′″ in Formula III is H, —OH, —OR^(a), halogen, (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, —NH₂, —NH(C₁-C₆)alkyl, —N[(C₁-C₆)alkyl]₂, —CN,(C₃-C₈)heteroaryl, (C₃-C₈)heterocycloalkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl, (C₃-C₈)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₈)heteroaryl-(C₁-C₆)alkylene-, (C₃-C₈)aryl(C₁-C₆)alkylene-, or(C₁-C₆)alkyl-(C₃-C₈)arylene.

Subscripts l, m, n, p and q independently are integers between 0 and 2inclusive, and at least one of l, m, n, or p is not 0.

in Formula IIII represents the option of having one or more doublebonds. For Formula III compounds, R^(a) is H, straight or branched chain(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₁₄)aryl,(C₃-C₁₄)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₁₄)aryl(C₁-C₆)alkylene-.

Any alkyl, alkylene, alkenylene, aryl, heteroaryl, cycloalkyl, orheterocycloalkyl in a Formula IIII compound is optionally substitutedwith one or more of halogen, oxo, —COOH, —CN, —NO₂, —OH, —NR^(d)R^(e),(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl,(C₃-C₈)aryl, (C₃-C₈)heteroaryl, (C₃-C₈)heterocycloalkyl, or(C₃-C₈)aryloxy; with R^(d) and R^(e) each independently being H,straight or branched (C₁-C₆)alkyl, optionally substituted (C₃-C₈)aryl,optionally substituted (C₃-C₁₄)aryl(C₁-C₆)alkylene-, orH₂N(C₁-C₆)alkylene-.

According to one embodiment substituent X in Formula III is N andsubstituents T and R are each independently —S(O)₂— and Q′ is(C₁-C₆)alkyl. Alternatively, substituent X is —CH— and each of Q′, R andT are independently —O—(CH₂)_(x)—O—, —OC(O)— or (CH₂)_(x)—OC(O)—.

Exemplary Formula III compounds include

The present invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of a Formulae I, II, orIII compound, or a pharmaceutically acceptable salt thereof; and apharmaceutically acceptable carrier.

According to another embodiment is provided a method for treatingmultiple myeloma or osteoporosis in a subject by modulating the activityof a cannabinoid receptor-2 (CB2), comprising administering to thesubject a therapeutically effective amount of a compound of Formulae I,II or III. Also provided is a method for modulating the activity of acannabinoid receptor-2 (CB2) by contacting a CB2 receptor with acompound according to Formulae I, II, or III.

In one embodiment of the invention is provided a compound according toFormula IV or a pharmaceutically acceptable salt thereof:

According to Formula IV R¹ and R² are independently H, alkyl, oralkenyl. R³ is alkyl, alkenyl, aryl, aralkyl, aralkenyl, heterocyclyl,heterocyclylalkyl, heteroaryl, or heteroarylalkyl.

Each of R⁴ and R⁵ in Formula IV are independently a bond, alkylenyl, oralkenylenyl. For Formula IV compounds each R⁶ and R⁷ are independentlyOH, F, Cl, Br, I, (C₁-C₆)alkyl, alkoxy, amino, COOH, CONH₂, SO₃H, PO₃H₂,CN, SH, NO₂ or CF₃. Subscripts p and q are independently 0, 1, 2, 3, 4,or 5. According to Formula IV, when R¹ and R² are both H, R⁴ and R⁵ areboth methylene, and p and q are 0, then R³ is not 4-dimethylaminophenyl.

The present invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of FormulaIV and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Cannabinoid receptor expression in human cancer cells. (A) Theprotein levels of CB1 and CB2 in MM cells (U266, MM.1S) and non-MM cells(pancreatic cancer panc2.03 9, ovarian cancer OVCAR3 and SKOV3) weredetermined by Western blot. Cells in logarithmic phase were harvestedand lysed by RIPA buffer containing protease cocktail inhibitors. Tojustify the expression difference of CB1 and CB2, mouse brain tissuelysate was used for the specific expression of CB 1. (B) CB2 expressionin various human MM cell lines and primary CD138+ MM cells. Per thesuggestion of the antibody's manufacturer, the human Jurkat cell lysatewas used as the positive control for CB2 expression. (C) CB2 mRNA levelwas measured by RT-PCR in the indicated cell lines, using CB2 specificprimers. GAPDH abundance was employed as the loading controls forimmunoblot and PCR assays.

FIG. 2. Inhibitory effects of CB2 ligands on human MM cell growth. HumanMM cell U266 (3×10⁴ cells per well in 96-well plate) was exposed to theknown CB2 ligands (A) or PAM (B) at indicated concentrations (0-10μmol/L) for 48 h. Cell proliferation was measured by [³H]-thymidineuptake assay as described in Materials and Methods. (C) U266 cells(3×10⁴ per well in 96-well plate) were treated with PAM at indicateddoses for 48 h. Cell viability and the viable cell number per well weredetermined using trypan blue exclusion assay. (D) Human chemoresistantmyeloma cell lines MM.1R (dexamethasone), RPMI-8226/LR5 (melphalan) andtheir respective parent cells MM.1S and RPMI-8226 were exposed to PAMfor 48 h. Cell proliferation was measured by ³H-thymidine incorporation.The data presented are the mean±SD of at least 3 independentmeasurements. (E) [S³⁵]-GTPγS binding assay of different ligands onhuman CB2 receptor expressed in CHO cells.

FIG. 3. Inhibitory activity of PAM (compound (1)) in CB2 knockdown oragonist treated cells. (A) CB2 gene silencing in MM1.S cells confirmedby Western blot with CB2antibody. (B) Human MM1.S cells expressing shRNAvector or expressing the specific shRNA against human CB2 were treatedwith vehicle control (white column), or the indicated concentrations ofPAM (black column) for 48 h. Cell proliferation was measured by [³H]TdRuptake. (C) and (D) Human U266 cells (4×10⁵ cells/well, 24 well plate)were treated with PAM in the absence or presence of CB2 agonistWin55212-2, or CP55940 for 48 h. The cell viability and the viablecells/well were determined using trypan blue exclusion assay.

FIG. 4. Effects of XIE35 and compound (1) on RANKL-induced osteoclastformation in bone marrow mononuclear cells (MNC). Upper panel: Murinebone marrow MNCs (1×10⁵ per well) were isolated by separation onFicoll-Hypaque gradients as described previously and cultured in thepresence of rmRANKL (12.5 ng/mL) plus rhM-CSF (10 ng/ml), and drugvehicle, XIE35 (1 μM) or compound (1) (1 μM) was added to appropriatewells. Half-medium change was performed every other day usingdrug-containing media where appropriate. After 5 days, the cells werefixed and stained for TRAP activity using a TRAP-staining kit(Sigma-Aldrich) according to the manufacturer's instructions. Lowerpanel: Human bone marrow MNCs (1×10⁵ per well) were treated with rhRANKL(50 ng/ml) plus rhM-CSF (10 ng/ml), and the respective compounds asdescribed above. After three weeks, differentiation into OCLs wasassessed by staining with monoclonal antibody 23c6 using aVectastatin-ABC-AP kit (Vector Laboratories). The antibody 23c6, whichrecognizes CD51/61 dimer constituting the OCL vitronectin receptor, waskindly provided by the Bone Biology Center of our university. Imageswere obtained with an Olympus IX70 microscope. Arrows designate thetypical multinucleated osteoclasts with 3 or more nuclei.

FIG. 5. Anti-osteoclastogenesis activity of exemplary compounds. (A)Compounds 61, 84 and 93 inhibit RANKL-induced osteoclastogenesis in adose-dependent manner. RAW 264.7 cells (3×10³ cells/well) were treatedwith or without RANKL (15 ng/mL), followed by addition of the indicatedconcentrations of 61, 84 and 93 for 5 days and stained for TRAPexpression. The data are the mean of three experiments carried out intriplicate. The bar indicates the SD. (B) Photographs of cells in thetest of compound 93 (original magnification 100×).

DETAILED DESCRIPTION

Definitions

For the purposes of this disclosure and unless otherwise specified, “a”or “an” means “one or more.”

“CB” is an abbreviation for “cannabinoid receptor.”

“CB1” is an abbreviation for “cannabinoid receptor subtype-1.”

“CB2” is an abbreviation for “cannabinoid receptor subtype-2.”

Generally, reference to a certain element such as hydrogen or H is meantto include all isotopes of that element. For example, if an R group isdefined to include hydrogen or H, it also includes deuterium andtritium. Hence, isotopically labeled compounds are within the scope ofthe invention.

In general, “substituted” refers to an organic group as defined below(e.g., an alkyl group) in which one or more bonds to a hydrogen atomcontained therein are replaced by a bond to non-hydrogen or non-carbonatoms. Substituted groups also include groups in which one or more bondsto a carbon(s) or hydrogen(s) atom are replaced by one or more bonds,including double or triple bonds, to a heteroatom. Thus, a substitutedgroup will be substituted with one or more substituents, unlessotherwise specified. In some embodiments, a substituted group issubstituted with 1, 2, 3, 4, 5, or 6 substituents. Examples ofsubstituent groups include: halogens (i.e., F, Cl, Br, and I);hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy,heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo);carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines;aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls;sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones;azides; amides; ureas; amidines; guanidines; enamines; imides;isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitrogroups; nitriles (i.e., CN); and the like.

Substituted ring groups such as substituted cycloalkyl, aryl,heterocyclyl and heteroaryl groups also include rings and fused ringsystems in which a bond to a hydrogen atom is replaced with a bond to acarbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl andheteroaryl groups may also be substituted with substituted orunsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

Alkyl groups include straight chain and branched alkyl groups havingfrom 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or,in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkylgroups further include cycloalkyl groups as defined below. Examples ofstraight chain alkyl groups include those with from 1 to 8 carbon atomssuch as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl,and n-octyl groups. Examples of branched alkyl groups include, but arenot limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl,isopentyl, and 2,2-dimethylpropyl groups. Representative substitutedalkyl groups may be substituted one or more times with substituents suchas those listed above.

Cycloalkyl groups are cyclic alkyl groups such as, but not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl groups. In some embodiments, the cycloalkyl group has 3 to 8ring members, whereas in other embodiments the number of ring carbonatoms range from 3 to 5, 3 to 6, or 3 to 7. Cycloalkyl groups furtherinclude mono-, bicyclic and polycyclic ring systems, such as, forexample bridged cycloalkyl groups as described below, and fused rings,such as, but not limited to, decalinyl, and the like. In someembodiments, polycyclic cycloalkyl groups have three rings. Substitutedcycloalkyl groups may be substituted one or more times with,non-hydrogen and non-carbon groups as defined above. However,substituted cycloalkyl groups also include rings that are substitutedwith straight or branched chain alkyl groups as defined above.Representative substituted cycloalkyl groups may be mono-substituted orsubstituted more than once, such as, but not limited to, 2,2-, 2,3-,2,4-2,5- or 2,6-disubstituted cyclohexyl groups, which may besubstituted with substituents such as those listed above.

Bridged cycloalkyl groups are cycloalkyl groups in which two or morehydrogen atoms are replaced by an alkylene bridge, wherein the bridgecan contain 2 to 6 carbon atoms if two hydrogen atoms are located on thesame carbon atom, or 1 to 5 carbon atoms, if the two hydrogen atoms arelocated on adjacent carbon atoms, or 2 to 4 carbon atoms if the twohydrogen atoms are located on carbon atoms separated by 1 or 2 carbonatoms. Bridged cycloalkyl groups can be bicyclic, such as, for examplebicyclo[2.1.1]hexane, or tricyclic, such as, for example, adamantyl.Representative bridged cycloalkyl groups include bicyclo[2.1.1]hexyl,bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl,bicyclo[3.2.2]nonyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2]decanyl,adamantyl, noradamantyl, bornyl, or norbornyl groups. Substitutedbridged cycloalkyl groups may be substituted one or more times withnon-hydrogen and non-carbon groups as defined above. Representativesubstituted bridged cycloalkyl groups may be mono-substituted orsubstituted more than once, such as, but not limited to, mono-, di- ortri-substituted adamantyl groups, which may be substituted withsubstituents such as those listed above.

Cycloalkylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to acycloalkyl group as defined above. In some embodiments, cycloalkylalkylgroups have from 4 to 20 carbon atoms, 4 to 16 carbon atoms, andtypically 4 to 10 carbon atoms. Substituted cycloalkylalkyl groups maybe substituted at the alkyl, the cycloalkyl or both the alkyl andcycloalkyl portions of the group. Representative substitutedcycloalkylalkyl groups may be mono-substituted or substituted more thanonce, such as, but not limited to, mono-, di- or tri-substituted withsubstituents such as those listed above.

Alkenyl groups include straight and branched chain and cycloalkyl groupsas defined above, except that at least one double bond exists betweentwo carbon atoms. Thus, alkenyl groups have from 2 to about 20 carbonatoms, and typically from 2 to 12 carbons or, in some embodiments, from2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, alkenylgroups include cycloalkenyl groups having from 4 to 20 carbon atoms, 5to 20 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbonatoms. Examples include, but are not limited to vinyl, allyl,—CH═CH(CH₃), —CH═C(CH₃)₂, —C(CH₃)═CH₂, —C(CH₃)═CH(CH₃), —C(CH₂CH₃)═CH₂,cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl,and hexadienyl, among others. Representative substituted alkenyl groupsmay be mono-substituted or substituted more than once, such as, but notlimited to, mono-, di- or tri-substituted with substituents such asthose listed above.

Cycloalkenylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of the alkyl group is replaced with a bond to acycloalkenyl group as defined above. Substituted cycloalkylalkenylgroups may be substituted at the alkyl, the cycloalkenyl or both thealkyl and cycloalkenyl portions of the group. Representative substitutedcycloalkenylalkyl groups may be substituted one or more times withsubstituents such as those listed above.

Alkynyl groups include straight and branched chain alkyl groups, exceptthat at least one triple bond exists between two carbon atoms. Thus,alkynyl groups have from 2 to about 20 carbon atoms, and typically from2 to 12 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4carbon atoms. Examples include, but are not limited to —C≡CH, —C≡C(CH₃),—C≡C(CH₂CH₃), —CH₂C≡CH, —CH₂C≡C(CH₃), and —CH₂C≡C(CH₂CH₃), among others.Representative substituted alkynyl groups may be mono-substituted orsubstituted more than once, such as, but not limited to, mono-, di- ortri-substituted with substituents such as those listed above.

Aryl groups are cyclic aromatic hydrocarbons that do not containheteroatoms. Aryl groups include monocyclic, bicyclic and polycyclicring systems. Thus, aryl groups include, but are not limited to, phenyl,azulenyl, heptalenyl, biphenylenyl, indacenyl, fluorenyl, phenanthrenyl,triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenyl, anthracenyl,indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments,aryl groups contain 6-14 carbons, and in others from 6 to 12 or even6-10 carbon atoms in the ring portions of the groups. Although thephrase “aryl groups” includes groups containing fused rings, such asfused aromatic-aliphatic ring systems (e.g., indanyl,tetrahydronaphthyl, and the like), it does not include aryl groups thathave other groups, such as alkyl or halo groups, bonded to one of thering members. Rather, groups such as tolyl are referred to assubstituted aryl groups. Representative substituted aryl groups may bemono-substituted or substituted more than once. For example,monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-,5-, or 6-substituted phenyl or naphthyl groups, which may be substitutedwith substituents such as those listed above.

Aralkyl groups are alkyl groups as defined above in which a hydrogen orcarbon bond of an alkyl group is replaced with a bond to an aryl groupas defined above. In some embodiments, aralkyl groups contain 7 to 20carbon atoms, 7 to 14 carbon atoms or 7 to 10 carbon atoms. Substitutedaralkyl groups may be substituted at the alkyl, the aryl or both thealkyl and aryl portions of the group. Representative aralkyl groupsinclude but are not limited to benzyl and phenethyl groups and fused(cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Representativesubstituted aralkyl groups may be substituted one or more times withsubstituents such as those listed above.

Heterocyclyl groups include aromatic (also referred to as heteroaryl)and non-aromatic ring compounds containing 3 or more ring members, ofwhich one or more is a heteroatom such as, but not limited to, N, O, andS. In some embodiments, heterocyclyl groups include 3 to 20 ringmembers, whereas other such groups have 3 to 6, 3 to 10, 3 to 12, or 3to 15 ring members. Heterocyclyl groups encompass unsaturated, partiallysaturated and saturated ring systems, such as, for example, imidazolyl,imidazolinyl and imidazolidinyl groups. The phrase “heterocyclyl group”includes fused ring species including those comprising fused aromaticand non-aromatic groups, such as, for example, benzotriazolyl,2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase alsoincludes bridged polycyclic ring systems containing a heteroatom suchas, but not limited to, quinuclidyl. However, the phrase does notinclude heterocyclyl groups that have other groups, such as alkyl, oxoor halo groups, bonded to one of the ring members. Rather, these arereferred to as “substituted heterocyclyl groups”. Heterocyclyl groupsinclude, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl,tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl,imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl,thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl,thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane,dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl,pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl,dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl,isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl,benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl,benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl,benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl,benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl(azabenzimidazolyl), triazolopyridyl, isoxazolopyridyl, purinyl,xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl,quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl,pteridinyl, thianaphthalenyl, dihydrobenzothiazinyl,dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl,tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl,tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl,tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl,tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups.Representative substituted heterocyclyl groups may be mono-substitutedor substituted more than once, such as, but not limited to, pyridyl ormorpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, ordisubstituted with various substituents such as those listed above.

Heteroaryl groups are aromatic ring compounds containing 5 or more ringmembers, of which, one or more is a heteroatom such as, but not limitedto, N, O, and S. Heteroaryl groups include, but are not limited to,groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, thiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl,thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl(pyrrolopyridyl), indazolyl, benzimidazolyl, imidazopyridyl(azabenzimidazolyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl,benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridyl,isoxazolopyridyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl,guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl,and quinazolinyl groups. Although the phrase “heteroaryl groups”includes fused ring compounds such as indolyl and 2,3-dihydro indolyl,the phrase does not include heteroaryl groups that have other groupsbonded to one of the ring members, such as alkyl groups. Rather,heteroaryl groups with such substitution are referred to as “substitutedheteroaryl groups.” Representative substituted heteroaryl groups may besubstituted one or more times with various substituents such as thoselisted above.

Heterocyclylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheterocyclyl group as defined above. Substituted heterocyclylalkylgroups may be substituted at the alkyl, the heterocyclyl or both thealkyl and heterocyclyl portions of the group. Representativeheterocyclyl alkyl groups include, but are not limited to,4-ethyl-morpholinyl, 4-propylmorpholinyl, furan-2-yl methyl, furan-3-ylmethyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-ylpropyl. Representative substituted heterocyclylalkyl groups may besubstituted one or more times with substituents such as those listedabove.

Heteroaralkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheteroaryl group as defined above. Substituted heteroaralkyl groups maybe substituted at the alkyl, the heteroaryl or both the alkyl andheteroaryl portions of the group. Representative substitutedheteroaralkyl groups may be substituted one or more times withsubstituents such as those listed above.

Groups described herein having two or more points of attachment (i.e.,divalent, trivalent, or polyvalent) within the compound of the inventionare designated by use of the suffix, “ene.” For example, divalent alkylgroups are alkylene groups, divalent aryl groups are arylene groups,divalent heteroaryl groups are divalent heteroarylene groups, and soforth. Substituted groups having a single point of attachment to thecompound of the invention are not referred to using the “ene”designation. Thus, e.g., chloroethyl is not referred to herein aschloroethylene.

Alkoxy groups are hydroxyl groups (—OH) in which the bond to thehydrogen atom is replaced by a bond to a carbon atom of a substituted orunsubstituted alkyl group as defined above. Examples of linear alkoxygroups include but are not limited to methoxy, ethoxy, propoxy, butoxy,pentoxy, hexoxy, and the like. Examples of branched alkoxy groupsinclude but are not limited to isopropoxy, sec-butoxy, tert-butoxy,isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groupsinclude but are not limited to cyclopropyloxy, cyclobutyloxy,cyclopentyloxy, cyclohexyloxy, and the like. Representative substitutedalkoxy groups may be substituted one or more times with substituentssuch as those listed above.

The terms “aryloxy” and “arylalkoxy” refer to, respectively, asubstituted or unsubstituted aryl group bonded to an oxygen atom and asubstituted or unsubstituted aralkyl group bonded to the oxygen atom atthe alkyl. Examples include but are not limited to phenoxy, naphthyloxy,and benzyloxy. Representative substituted aryloxy and arylalkoxy groupsmay be substituted one or more times with substituents such as thoselisted above.

The term “amine” (or “amino”) as used herein refers to —NHR⁴ and —NR⁵R⁶groups, wherein R⁴, R⁵ and R⁶ are independently hydrogen, or asubstituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl,aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. Insome embodiments, the amine is NH₂, methylamino, dimethylamino,ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, orbenzylamino.

The term “amide” refers to a —NR′R″C(O)— group wherein R′ and R″ eachindependently refer to a hydrogen, (C₁-C₈)alkyl, or (C₃-C₆)aryl.

The term ‘nitrile or cyano can be used interchangeably and refer to a—CN group which is bound to a carbon atom of a heteroaryl ring, arylring and a heterocycloalkyl ring.

The substituent —CO₂H, may be replaced with bioisosteric replacementssuch as:

and the like, wherein R has the same definition as R′ and R″ as definedherein. See, e.g., THE PRACTICE OF MEDICINAL CHEMISTRY (Academic Press:New York, 1996), at page 203.

Those of skill in the art will appreciate that compounds of theinvention may exhibit the phenomena of tautomerism, conformationalisomerism, geometric isomerism and/or optical isomerism. As the formuladrawings within the specification and claims can represent only one ofthe possible tautomeric, conformational isomeric, optical isomeric orgeometric isomeric forms, it should be understood that the inventionencompasses any tautomeric, conformational isomeric, optical isomericand/or geometric isomeric forms of the compounds having one or more ofthe utilities described herein, as well as mixtures of these variousdifferent forms.

“Tautomers” refers to isomeric forms of a compound that are inequilibrium with each other. The concentrations of the isomeric formswill depend on the environment the compound is found in and may bedifferent depending upon, for example, whether the compound is a solidor is in an organic or aqueous solution. For example, in aqueoussolution, pyrazoles may exhibit the following isomeric forms, which arereferred to as tautomers of each other:

As readily understood by one skilled in the art, a wide variety offunctional groups and other structures may exhibit tautomerism, and alltautomers of compounds as described herein are within the scope of thepresent invention.

Stereoisomers of compounds, also known as “optical isomers,” include allchiral, diastereomeric, and racemic forms of a structure, unless thespecific stereochemistry is expressly indicated. Thus, compounds used inthe present invention include enriched or resolved optical isomers atany or all asymmetric atoms as are apparent from the depictions. Bothracemic and diastereomeric mixtures, as well as the individual opticalisomers can be isolated or synthesized so as to be substantially free oftheir enantiomeric or diastereomeric partners, and these are all withinthe scope of the invention.

“A pharmaceutically acceptable carrier” is a phrase that denotes acarrier such as but not limited to a diluent, an excipient, a wettingagent, a buffering agent, a suspending agent, a lubricating agent, anadjuvant, a vehicle, a delivery system, an emulsifier, a disintegrant,an absorbent, a preservative, a surfactant, a colorant, a flavorant, asweetener, or a mixture of any two or more thereof. Pharmaceuticallyacceptable excipients and carriers are generally known and, hence, areincluded in the instant invention. Such materials are described, forexample, in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21^(st)ed., The University of Philadelphia (2005).

Pharmaceutical compositions and medicaments may be prepared by mixingone or more compounds of the invention, prodrugs thereof,pharmaceutically acceptable salts thereof, stereoisomers thereof,tautomers thereof, or solvates thereof, with pharmaceutically acceptablecarriers, excipients, binders, diluents or the like to prevent and treatdisorders associated with cannabinoid receptors. The compounds andcompositions of the invention may be used to prepare formulations andmedicaments that prevent or treat a variety of disorders associated withcannabinoid receptors, and described herein. For example, disorders anddiseases such as obesity, smoking addiction, cardimetabolic riskfactors, and other disorder and diseases associated with the centralnervous system. Such compositions can be in the form of, for example,granules, powders, tablets, capsules, syrup, suppositories, injections,emulsions, elixirs, suspensions or solutions. The instant compositionscan be formulated for various routes of administration, for example, byoral, parenteral, topical, rectal, nasal, vaginal administration, or viaimplanted reservoir. Parenteral or systemic administration includes, butis not limited to, subcutaneous, intravenous, intraperitoneally,intramuscular, intra-articular, intrasynovial, intrasternal,intrathecal, intralesional and intracranial injections. The followingdosage forms are given by way of example and should not be construed aslimiting the instant invention.

Pharmaceutically acceptable salts of the invention compounds areconsidered within the scope of the present invention. The compound ofthe invention has a number of basic nitrogen groups, and as such,pharmaceutically acceptable salts can be formed with inorganic acids(such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid,and phosphoric acid), organic acids (e.g. formic acid, acetic acid,trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, lacticacid, maleic acid, citric acid, succinic acid, malic acid,methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid)or acidic amino acids (such as aspartic acid and glutamic acid). Thecompounds of the present invention may have acidic substituent groups,and in such cases, it can form salts with metals, such as alkali andearth alkali metals (e.g. Na⁺, Li⁺, K⁺, Ca²⁺, Mg²⁺, Zn²⁺), organicamines (e.g. ammonia, trimethylamine, triethylamine, pyridine, picoline,ethanolamine, diethanolamine, triethanolamine) or basic amino acids(e.g. arginine, lysine and ornithine).

Certain compounds within the scope of the invention are derivativesreferred to as prodrugs. The expression “prodrug” denotes a derivativeof a known direct acting drug, e.g. esters and amides, which derivativehas enhanced delivery characteristics and therapeutic value as comparedto the drug, and is transformed into the active drug by an enzymatic orchemical process; see Notari, R. E., “Theory and Practice of ProdrugKinetics,” Methods in Enzymology 112: 309-23 (1985); Bodor, N., “NovelApproaches in Prodrug Design,” Drugs of the Future 6: 165-82 (1981); andBundgaard, H., “Design of Prodrugs: Bioreversible-Derivatives forVarious Functional Groups and Chemical Entities,” in DESIGN OF PRODRUGS(H. Bundgaard, ed.), Elsevier (1985), Goodman and Gilmans, THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, 8th ed., McGraw-Hill (1992).

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, and caplets are acceptableas solid dosage forms. These can be prepared, for example, by mixing oneor more compounds of the instant invention, or pharmaceuticallyacceptable salts or tautomers thereof, with at least one additive suchas a starch or other additive. Suitable additives are sucrose, lactose,cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates,chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins,collagens, casein, albumin, synthetic or semi-synthetic polymers orglycerides. Optionally, oral dosage forms can contain other ingredientsto aid in administration, such as an inactive diluent, or lubricantssuch as magnesium stearate, or preservatives such as paraben or sorbicacid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, adisintegrating agent, binders, thickeners, buffers, sweeteners,flavoring agents or perfuming agents. Tablets and pills may be furthertreated with suitable coating materials known in the art.

Compounds

The compounds represented by Formulae I, I′, II, III, III′, and VI(below) are potent modulators of cannabinoid receptor-2 (CB2). As statedabove, the endocannabinoid system, particularly CB2, plays an importantrole in various physiological processes, including regulation ofskeletal remodeling and bone mass. These physiological processes areimplicated to play a role in the development and progression ofosteoporosis. While the physiological role of endocannabinoid system inmultiple myeloma (MM), is not clear, cell based studies using FormulaeI, I′, II, III, III′, and IV compounds have revealed an anti-MMactivity.

In one aspect, compounds represented by Formula I, II, and III, or theirpharmaceutically acceptable salts are provided:

For Formula I compounds D is H, —OH, —OR^(a), (C₁-C₆)alkyl or

In one embodiment, D is

and is an aromatic phenyl group that can be optionally substituted byone, two, or three substituent groups.

represents the point of attachment of the ring to the Formula Iscaffold. According to Formula I, D can be an —OR^(a), or a (C₁-C₆)alkylgroup. When D is OR^(a), however, R^(a) is selected from the groupconsisting of hydrogen, straight or branched chain (C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C₃-C₁₄)aryl,(C₃-C₁₄)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₁₄)aryl(C₁-C₆)alkylene-.According to the above formulas, A, B and Q in Formula I are eachindependently (C₁-C₆)alkylene, (C₂-C₆)alkenylene and (C₂-C₆)alkynylene,while subscripts e, f and g independently are integers between 0 and 6inclusive.

For compounds that conform to Formula I, substituent groups V, W, X, Y,or Z are each independently a bond, —C(R′″)₂—, —CR′″—, —NR′″—, —N—, —O—,—C(O)—, and —S—. However, no two adjacent members of V, W, X, Y, or Z inFormula I are simultaneously —O—, —S—, or —NR′″—. According to Formula Icompounds, V, W, X, Y, and Z are each independently a bond, —C(R′″)₂—,—CR′″—, —NR′″—, —N—, —O—, —C(O)—, or —S—. Compounds according to FormulaI encompass, therefore, species in which one, two or each of the threering structures represents a heteroaryl group, a heterocycle group, acycloalkyl group or an aryl ring. Exemplary of heteroaryl andheterocycle rings are oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,oxepanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, 1,3-dioxanyl,oxazolidinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepinyl,piperazinyl, morpholinyl, tetrahydrothiopyranyl-1-oxide,tetrahydrothiopyranyl-1,1-dioxide, pyrrolidinonyl, piperidinonyl,azepinonyl, piperazidinonyl, oxazidilinonyl, azetidinonyl, ormorpholinonyl. When any of V, W, X, Y, or Z is a —C(R′″)₂—, —CR′″—,—NR′″— group, substituent R′″ is H, OH, OR^(a), halogen, (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, —NH₂, —NH(C₁-C₆)alkyl, —N[(C₁-C₆)alkyl]₂, —CN,(C₃-C₈)heteroaryl, (C₃-C₈)heterocycloalkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl, (C₃-C₈)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₈)heteroaryl-(C₁-C₆)alkylene-, (C₃-C₈)aryl(C₁-C₆)alkylene-,(C₃-C₈)aryl(C₁-C₆)alkenylene-, or (C₁-C₆)alkyl-(C₃-C₈)arylene.

Subscripts l, m, n, p and q independently are integers between 0 and 2inclusive and at least one of l, m, n, p, or q is not zero (0). Toaccommodate for the presence of aromatic and non-aromatic ring systems,Formula I recites

to represent the option of having one or more double bonds within a ringsystem. Further, for compounds that conform to Formula I, any alkyl,alkylene, alkenylene, aryl, heteroaryl, cycloalkyl, or heterocycloalkylis optionally substituted with one or more halogen, oxo, —COOH, —CN,—NO₂, —OH, —NR^(d)R^(e), (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy,(C₁-C₆)haloalkyl, (C₃-C₈)aryl, (C₃-C₈)heteroaryl,(C₃-C₈)heterocycloalkyl, or (C₃-C₈)aryloxy; with each of R^(d) and R^(e)being independently H, straight or branched (C₁-C₆)alkyl, optionallysubstituted (C₃-C₈)aryl, optionally substituted(C₃-C14)aryl(C₁-C₆)alkylene-, or H₂N(C₁-C₆)alkylene-. In one embodiment,the compound of Formula I is subject to the proviso that where each

are independently phenyl, B_(f) and Q_(g) are both methylene, andsubscript e is 0, D is a 4-dimethylaminophenyl. However, the methods oftreatment described below are not necessarily subject to this proviso.

In one aspect, the compounds of Formula I also include the compounds ofFormula IV:

For Formula IV compounds, R¹ and R² are independently H, alkyl, oralkenyl and substituent R³ can be alkyl, alkenyl, aryl, aralkyl,aralkenyl, heterocyclyl, heterocyclylalkyl, heteroaryl, orheteroarylalkyl. In one embodiment of Formula IV, R⁴ and R⁵ areindependently a bond, alkylenyl, or alkenylenyl and each R⁶ and R⁷ isindependently H, OH, F, Cl, Br, I, alkoxy, amino, —COOH, —C(O)NH₂, SO₃H,PO₃H₂, —CN, —SH, —NO₂, or CF₃ groups. For Formula IV compounds,subscripts p and q are independently 0, 1, 2, 3, 4, or 5. In someembodiments, the compounds of Formula IV are subject to the proviso thatwhen R¹ and R² are both H, R⁴ and R⁵ are both methylene, and subscriptsp and q are 0, R³ in Formula IA is not 4-dimethylaminophenyl.

In some embodiments of Formula IV, R³ is a substituted phenyl ringbelonging to Formula B, with each R⁸ being independently selected fromOH, F, Cl, Br, I, (C₁-C₆)alkyl, alkoxy, amino, —COOH, —C(O)NH₂, SO₃H,PO₃H₂, —CN, —SH, —NO₂, or CF₃ and t is 0, 1, 2, 3, 4 or 5.

In one embodiment of Formula IV, R¹ and R² are independently a H or aC₁-C₈ alkyl group, substituent R³ is alkyl, aryl, or aralkyl, wherealkyl has from 1-8 carbons and substituent groups R⁴ and R⁵ areindependently a bond, C₁-C₈ alkylenyl, or C₁-C₈ alkenylenyl. Accordingto this embodiment, each R⁶ and R⁷ is independently OH, F, Cl, Br, I,C₁-C₈ alkoxy, or amino, subscripts p, q, and t are independently 0, 1,or 2 and each R⁸ is independently OH, F, Cl, Br, I, alkoxy, amino.

For certain Formula IV compounds, R¹ and R² independently are H, R⁴ andR⁵ are both a bond, methylene, or ethylene and R³ is

For these compounds each R⁸ is independently OH, F, Cl, Br, I, methoxy,trifluoromethoxy, NH₂, dimethylamino, or diethylamino group and p, q,and t are independently 0, 1, or 2.

Exemplary Formula I compounds without limitation are those shown belowin Table 1:

TABLE 1

As described generally above, the invention also provides compoundsaccording to Formula I′:

In some embodiments, the Formula I′ compound is one where D is H; D′ isphenyl; B and Q are independently (C₁-C₆)alkylene; e is 0 and each offand g is 1; and each of R^(a′), R^(a″). and R^(a′″) is independentlyselected from the group consisting of H, and straight or branched chain(C₁-C₆)alkyl.

Exemplary Formula I′ compounds include those in the following table:

In another embodiment, is provided a CB2 ligand that conforms to FormulaII.

For Formula II compounds or their pharmaceutically acceptable salts,substituent groups A, B′ and E are each independently a bond, —C(R′″)₂—,—CR′″—, —NR′″—, —N—, —O—, —C(O)—, or —S—, however, no two adjacentmembers of A, B′ and E can simultaneously be —O—, —S—, or —NR′″—. Whilesubscripts h, j and k independently are integers between 0 and 2inclusive, at least one of h, j or k is not 0 in Formula II.

In one embodiment, D and D″ are each independently —C(O), —CH₂C(O)—,(C₁-C₆)alkylene, —C(O)NH—, or —NHC(O)—. Formula II, furthermore,prescribes compounds that have a fused ring system, with

representing the option of having a C₅ member or a C₆ member fused ringthat optionally has one or more double bonds.

For Formula II compounds, R and R₁ are each independently —OH, —OR^(a),(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,(C₃-C₈)heteroaryl, (C₃-C₈)heterocycloalkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl, (C₃-C₈)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₈)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₈)aryl(C₁-C₆)alkylene-.

When Group R and R₁ are —OR^(a), R^(a) is H, straight or branched chain(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₁₄)aryl,(C₃-C₁₄)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₁₄)aryl(C₁-C₆)alkylene-.

For compounds conforming to Formula II, any alkyl, alkylene, aryl,heteroaryl, cycloalkyl, or heterocycloalkyl is optionally substitutedwith one or more halogen, oxo, —COOH, —CN, —NO₂, —OH, —NR^(d)R^(e),(C₁-C₆)alkoxy, or (C₃-C₈)aryloxy; with each R^(d) and R^(e)independently being H, straight or branched (C₁-C₆)alkyl, optionallysubstituted (C₃-C₁₄)aryl(C₁-C₆)alkylene-, or H₂N(C₁-C₆)alkylene-.

According to one embodiment are provided Formula II compounds in which

represents an optionally substituted phenyl group, groups A and E areindependently —N—, —O—, or —S— and

represents the option of having one or more double bonds. As prescribed,therefore, compounds that conform to Formula II include withoutlimitation analogs of phenylimidazole, 3H-indazole, indole,benz[d]oxazole, or 2,3-dihydrobenzo[d]oxazole, benzo[d] thiazole.

Exemplary of Formula II compounds without limitation are the following:

Also provided by the present invention are modulators of the CB2receptor that conform to Formula III.

For Formula III compounds X is N, or —CH—, groups Q′, R and T are eachindependently (C₁-C₆)alkyl, —S(O)₂—, —S(O)—, —S(O)₂NHR″,—O—(CH₂)_(x)—O—, —OC(O)— or (CH₂)_(x)—OC(O)—, while groups G, H, J, L,or M are each independently a bond, —C(R′″)₂—, —CR′″—, —NR′″—, —N—, —O—,—C(O)—, or —S—.

For Formula III compounds, however, no two adjacent members of G, H, J,L, or M can simultaneously be —O—, —S—, or —NR′″—. According to oneembodiment, R′″ is H, OH, OR^(a), halogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,—NH₂—NH(C₁-C₆)alkyl, —N[(C₁-C₆)alkyl]₂, —CN, (C₃-C₈)heteroaryl,(C₃-C₈)heterocycloalkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl,(C₃-C₈)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₈)heteroaryl-(C₁-C₆)alkylene-, (C₃-C₈)aryl(C₁-C₆)alkylene-, or(C₁-C₆)alkyl-(C₃-C₈)arylene. The dashed circle,

represents the option of having one or more double bonds and for FormulaIII compounds, subscripts l, m, n, p and q independently are integersbetween 0 and 2 inclusive, with at least one of l, m, n, p, or q being anon-zero (0) integer.

When R′″ is −OR^(a), group R^(a) is hydrogen, straight or branched chain(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₁₄)aryl,(C₃-C₁₄)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₁₄)aryl(C₁-C₆)alkylene-.

Furthermore, for compounds conforming to Formula III, any alkyl,alkylene, alkenylene, aryl, heteroaryl, cycloalkyl, or heterocycloalkylis optionally substituted with one or more members selected from thegroup consisting of halogen, oxo, —COOH, —CN, —NO₂, —OH, —NR^(d)R^(e),(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl,(C₃-C₈)aryl, (C₃-C₈)heteroaryl, (C₃-C₈)heterocycloalkyl, or(C₃-C₈)aryloxy; with each R^(d) and R^(e) group being independently H,straight or branched (C₁-C₆)alkyl, optionally substituted (C₃-C₈)aryl,optionally substituted (C₃-C₁₄)aryl(C₁-C₆)alkylene-, orH₂N(C₁-C₆)alkylene-.

For certain compounds according to Formula III, substituent X is anitrogen, substituent groups T and R are each —S(O)₂— and Q′ is a(C₁-C₆)alkyl. For other Formula III compounds, X is —CH— and each of Q,R and T are independently —O—(CH₂)_(x)—O—, —OC(O)— or (CH₂)_(x)—OC(O)—.Illustrated below, without limitation are two compounds that conform toFormula III.

Pharmaceutical Compositions

Compounds of Formula I, II, III or IV can each be administered to apatient or subject in need of treatment either individually, or incombination with other therapeutic agents that have similar biologicalactivities. For example, Formulae I, II, III or IV compounds andcompositions can be administered as a single dose or as multiple dailydoses by a practicing medical practitioner. When combination therapy isused, however, the compound and the other therapeutic agent areadministered separately at different time intervals, or simultaneously.

Pharmaceutical formulations may include a compound I, II, III or IV, ora pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier. In some embodiments, the composition furthercontains, in accordance with accepted practices of pharmaceuticalcompounding, one or more additional therapeutic agents, pharmaceuticallyacceptable excipients, diluents, adjuvants, stabilizers, emulsifiers,preservatives, colorants, buffers, flavor imparting agents.

In one embodiment, the pharmaceutical composition includes a compoundaccording to Formula I or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

For Formula I compounds D is H, OH, OR^(a), (C₁-C₆)alkyl and

In one embodiment, D is

and is an aromatic phenyl group that can be optionally substituted byone, two, or three substituent groups.

represents the point of attachment of the ring to the Formula Iscaffold. According to Formula I, D can be an —OR^(a), or a (C₁-C₆)alkylgroup. When D is OR^(a), R^(a) is H, straight or branched chain(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₁₄)aryl,(C₃-C₁₄)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, or (C₃-C₁₄)aryl(C₁-C₆)alkylene-.Groups A, B and Q in Formula I are each independently (C₁-C₆)alkylene,(C₂-C₆)alkenylene or (C₂-C₆)alkynylene, while subscripts e, f and gindependently are integers between 0 and 6 inclusive. In Formula Icompounds, V, W, X, Y, and Z are each independently a bond, —C(R′″)₂—,—CR′″—, —NR′″—, —N—, —O—, —C(O)—, or —S—. However, no two adjacentmembers of V, W, X, Y, and Z in Formula I are simultaneously —O—, —S—,or —NR′″—. Compounds according to Formula I encompass, therefore,species in which one, two or each of the three ring structuresrepresents a heteroaryl group, a heterocycle group, a cycloalkyl groupor an aryl ring. Exemplary of heteroaryl and heterocycle rings areoxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepanyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, 1,3-dioxanyl, oxazolidinyl,azetidinyl, pyrrolidinyl, piperidinyl, azepinyl, piperazinyl,morpholinyl, tetrahydrothiopyranyl-1-oxide,tetrahydrothiopyranyl-1,1-dioxide, pyrrolidinonyl, piperidinonyl,azepinonyl, piperazidinonyl, oxazidilinonyl, azetidinonyl, andmorpholinonyl.

In one embodiment, the pharmaceutical formulation includes a compound ofFormula I, where any of V, W, X, Y, or Z is a —C(R′″)₂—, —CR′″—, —NR′″—group, substituent R′″ is —H, —OH, —OR^(a), halogen, (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, —NH₂, —NH(C₁-C₆)alkyl, —N[(C₁-C₆)alkyl]₂, —CN,(C₃-C₈)heteroaryl, (C₃-C₈)heterocycloalkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)aryl, (C₃-C₈)heterocycloalkyl-(C₁-C₆)alkylene-,(C₃-C₈)heteroaryl-(C₁-C₆)alkylene-, (C₃-C₈)aryl(C₁-C₆)alkylene-,(C₃-C₈)aryl(C₁-C₆)alkenylene-, or (C₁-C₆)alkyl-(C₃-C₈)arylene; l, m, n,p and q independently are integers between 0 and 2 inclusive, and atleast one of l, m, n, p, or q is not zero (0). To accommodate for thepresence of aromatic and non-aromatic ring systems, Formula I recites

to represent the option of having one or more double bonds within a ringsystem. Further, for compounds that conform to Formula I, any alkyl,alkylene, alkenylene, aryl, heteroaryl, cycloalkyl, or heterocycloalkylis optionally substituted with halogen, oxo, —COOH, —CN, —NO₂, —OH,—NR^(d)R^(e), (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy,(C₁-C₆)haloalkyl, (C₃-C₈)aryl, (C₃-C₈)heteroaryl,(C₃-C₈)heterocycloalkyl and (C₃-C₈)aryloxy; with each of R^(d) and R^(e)being H, straight or branched (C₁-C₆)alkyl, optionally substituted(C₃-C₈)aryl, optionally substituted (C₃-C₁₄)aryl(C₁-C₆)alkylene-, andH₂N(C₁-C₆)alkylene-.

In one embodiment, the pharmaceutical composition comprises a compoundof Formula IV, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier:

In such pharmaceutical compositions including a compound of Formula IV,R¹ and R² are independently H, alkyl, or alkenyl; R³ is alkyl, alkenyl,aryl, aralkyl, aralkenyl, heterocyclyl, heterocyclylalkyl, heteroaryl,heteroarylalkyl; R⁴ and R⁵ are independently a bond, alkylenyl, oralkenylenyl; each R⁶ and R⁷ is independently OH, F, Cl, Br, I,(C₁-C₆)alkyl, alkoxy, amino, —COOH, —C(O)NH₂, SO₃H, PO₃H₂, —CN, —SH,—NO₂, or CF₃; and p and q are independently 0, 1, 2, 3, 4, or 5.

In one embodiment, the pharmaceutical composition comprises a compoundselected from those illustrated in Table 1 or a pharmaceuticallyacceptable salt, and a pharmaceutically acceptable carrier.

Multiple Myeloma and/or Osteoporosis

It has been determined that the compounds of Formulae I, I′, II, III,III′ and IV are CB-2 receptor inverse agonists. A link is also herebyestablished between CB-2 receptors and the treatment of multiple myelomaand osteoporosis. By using two distinct CB2 antibodies that are able todistinguish active and inactive CB2 receptors, the present investorsfound that the CB2 receptors on human MM cells are in active form. Basedon the high levels of expression on MM cells and nature of the CB2receptor on human MM cells, it is expected that CB2 ligands withantagonistic activities can be employed to inhibit MM growth. Thus, byadministering a compound of Formula I, I′, II, III, III′ or IV to asubject, or by contacting a compound of Formula I, I′, II, III, III′ orIV with multiple myeloma cells or cells causing osteoporosis, theactivity of the CB-2 receptors may be modulated such that the cells areinactivated, their activity is substantially altered, or the subject istreated.

In one embodiment, a method for treating multiple myeloma orosteoporosis in a subject by modulating the activity of a cannabinoidreceptor-2 (CB2), includes administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula IV:

In such methods utilizing Formula IV, R¹ and R² are independently H,alkyl, or alkenyl; R³ is alkyl, alkenyl, aryl, aralkyl, aralkenyl,heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl; R⁴ and R⁵are independently a bond, alkylenyl, or alkenylenyl; each R⁶ and R⁷ isindependently OH, F, Cl, Br, I, (C₁-C₆)alkyl, alkoxy, amino, —COOH,—C(O)NH₂, SO₃H, PO₃H₂, —CN, —SH, —NO₂, CF₃; and p and q areindependently 0, 1, 2, 3, 4, or 5. In one embodiment, the treatment isfor multiple myeloma.

In another aspect, a method for modulating the activity of a cannabinoidreceptor-2 (CB2) in a subject, includes contacting the CB-2 receptorwith a compound of Formula IV:

In such methods utilizing Formula IV, R¹ and R² are independently H,alkyl, or alkenyl; R³ is alkyl, alkenyl, aryl, aralkyl, aralkenyl,heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl; R⁴ and R⁵are independently a bond, alkylenyl, or alkenylenyl; each R⁶ and R⁷ isindependently OH, F, Cl, Br, I, (C₁-C₆)alkyl, alkoxy, amino, —COOH,—C(O)NH₂, SO₃H, PO₃H₂, —CN, —SH, —NO₂, CF₃; and p and q areindependently 0, 1, 2, 3, 4, or 5.

In another aspect, a method is provided including modulating theactivity of a cannabinoid receptor-2 (CB2) in a subject suffering fromosteoporosis. In one embodiment, the modulating includes administering aCB-2 receptor inverse agonist to the subject. In one embodiment of themethods, the CB-2 receptor inverse agonist includes a compound ofFormula IV:

In such methods utilizing Formula IV, R¹ and R² are independently H,alkyl, or alkenyl; R³ is alkyl, alkenyl, aryl, aralkyl, aralkenyl,heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl; R⁴ and R⁵are independently a bond, alkylenyl, or alkenylenyl; each R⁶ and R⁷ isindependently OH, F, Cl, Br, I, (C₁-C₆)alkyl, alkoxy, amino, —COOH,—C(O)NH₂, SO₃H, PO₃H₂, —CN, —SH, —NO₂, CF₃; and p and q areindependently 0, 1, 2, 3, 4, or 5.

The compositions may be administered orally, or parenterally, the term“parenteral” as used herein includes subcutaneous injections,intravenous, intramuscular, intrasternal injection or infusiontechniques. Suitable oral compositions in accordance with the inventioninclude without limitation tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsion, hard or softcapsules, syrups or elixirs. The dosage form may be a single unit dosageform that includes any of the compounds of Formula I, I′, II, III, III′or IV, or a pharmaceutically salt thereof. Such dosage forms may includea pharmaceutically acceptable carrier.

Compositions for parenteral administrations are administered in asterile medium. Depending on the vehicle used and concentration theconcentration of the drug in the formulation, the parenteral formulationcan either be a suspension or a solution containing dissolved drug.Adjuvants such as local anesthetics, preservatives and buffering agentscan also be added to parenteral compositions.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

Synthesis of Compounds

General Synthetic Methodology

The preparation of substituted amide compounds used in the couplingreactions below were prepared via hydrolysis of corresponding2-phenylacetonitriles in concentrated H₂SO₄ (Scheme 1). The reactionswere typically conducted at about 0° C. for about 12 hours.

The compounds described below and shown in Table 1, were synthesized bythe method a or b of Scheme. In Scheme 1a, the coupling reaction betweenthe amide and aldehyde was performed in anhydrous dichloroethane (DCE)with the catalyst trimethylsilyl chloride (TMSCl) at about 70° C. for3-12 hours. In Scheme 1b, the coupling reaction was performed inanhydrous dichloromethane (DCM) with the catalyst F₃CSO₃SiMe₃, at roomtemperature for about 12 hours.

Chemistry

All reagents were purchased from commercial sources and used withoutfurther purification. Analytical thin-layer chromatography (TLC) wasperformed on SiO₂ plates on Alumina. Visualization was accomplished byUV irradiation at 254 nm. Preparative TLC was conducted usingPreparative Silica gel TLC plates (1000 μm, 20 cm×20 cm). Flash columnchromatography was performed using Biotage Isolera flash purificationsystem. ¹H NMR was recorded on a Bruker 400 MHz spectrometer. Chemicalshifts are reported as δ values in parts per million (ppm) as referencedto residual solvent. ¹H NMR spectra are tabulated as follows: chemicalshift, multiplicity (s=singlet, bs=broad singlet, d=doublet, t=triplet,q=quartet, m=multiplet), coupling constant(s), and number of protons.Flash column chromatography was performed using SiO₂ 60 (particle size0.040-0.055 mm, 230-400 mesh).

The chemical purity of the target compounds was >95% as determined usingthe following conditions: a Shimadzu HPLC-MS-MS with a HAMILTON reversedphase column (HxSil, C18, 3 μm, 2.1×50 mm (H2)); Eluent A: 5% CH₃CN inH₂O, eluent B: 90% CH₃CN in H₂O (Table 2); flow rate of 0.2 mL/min; UVdetector, 254 nm.

TABLE 2 HPLC-MS-MS Eluent Compositions T (min) A (%) B (%) 0.0 5 95 3.005 95 7.00 90 10 12.00 90 10 18.00 5 95 20.00 stop stop

Preparation of 2-Phenylacetamide

Benzyl cyanide (5 g, 42.7 mmol) was added slowly to concentratedsulfuric acid (20 ml) cooled by water-ice bath. The solution was stirredovernight. The reaction mixture was poured into ice water andneutralized with 20% NaOH. The aqueous phase was extracted by ethylacetate (3×15 mL). The combined organic layers were washed with water(3×10 mL) and brine (3×10 mL), dried over anhydrous MgSO₄, filtered, andconcentrated under reduced pressure. The residue was recrystallized fromethyl acetate and hexane to give the title compound (4.5 g, 78%). ¹H NMR(400 MHz, DMSO-d₆) δ 7.54 (s, 1H), 7.20-7.32 (m, 5H), 6.87 (s, 1H), 3.38(s, 2H).

2-(4-Chlorophenyl)acetamide

The same procedure was followed using 2-(4-chloro)benzyl cyanide. ¹H NMR(400 MHz, DMSO-d₆) δ 7.49 (s, 1H), 7.34-7.35 (m, 2H), 7.26-7.27 (m, 2H),6.92 (s, 1H), 3.34-3.37 (m, 2H).

(4-(Trifluoromethyl)phenyl)acetamide

The same procedure was followed using 4-trifluoromethylbenzyl cyanide.¹H NMR (400 MHz, DMSO-d₆) δ 7.65 (d, J=8.0 Hz, 2H), 7.58 (s, 1H), 7.49(d, J=8.0 Hz, 2H), 7.00 (s, 1H), 3.51 (s, 2H).

General Protocol for the Coupling Reaction Between Amide andAldehyde—Method 1.

N,N′-((4-(dimethylamino)phenyl)methylene)bis(2-phenylacetamide) (1). Toa suspension of 4-(dimethylamino)benzaldehyde (149 mg, 1 mmol) and2-phenylacetamide (270 mg, 2 mmol) in 2 mL anhydrous DCE, TMSCl (216 mg,2 mmol) was added. The mixture was heated at 70° C. for 12 h, thencooled to room temperature and the crude product precipitated from thesolution. The crude product was recrystallized with methanol and hexaneto give the final product (140 mg, 35%). ¹H NMR (400 MHz, DMSO-d₆) δ8.89 (d, J=8.0 Hz, 2H), 7.59 (s, 2H), 7.41 (d, J=8.8 Hz, 2H), 7.21-7.32(m, 10H), 6.54 (t, J=8.0 Hz, 1H), 3.52 (dd, J=14.0, 15.6 Hz, 4H), 3.06(s, 6H).

N,N′-(phenylmethylene)bis(2-phenylacetamide) (2). Yield: 67%. ¹H NMR(400 MHz, DMSO-d₆) δ 8.78 (d, J=7.2 Hz, 2H), 7.21-7.35 (m, 15H), 6.55(t, J=7.8 Hz, 1H), 3.50 (dd, J=13.8, 20.4 Hz, 4H).

N,N′-((2-fluorophenyl)methylene)bis(2-phenylacetamide) (3). Yield: 64%.¹H NMR (400 MHz, DMSO-d₆) δ 8.87 (d, J=7.8 Hz, 2H), 7.44 (t, J=7.8 Hz,1H), 7.36 (q, J=6.6 Hz, 1H), 7.17-7.29 (m, 12H), 6.74 (t, J=7.8 Hz, 1H),3.48 (dd, J=14.4, 24.0 Hz, 4H).

N,N′-((4-fluorophenyl)methylene)bis(2-phenylacetamide) (5). Yield: 72%.¹H NMR (400 MHz, DMSO-d₆) δ 8.79 (d, J=8.0 Hz, 2H), 7.16-7.36 (m, 14H),6.54 (t, J=8.0 Hz, 1H), 3.52 (dd, J=14.4, 15.6 Hz, 4H).

N,N′-((4-chlorophenyl)methylene)bis(2-phenylacetamide) (6). Yield: 71%.¹H NMR (400 MHz, DMSO-d₆) δ 8.83 (d, J=7.8 Hz, 2H), 7.41 (d, J=7.8 Hz,2H), 7.21-7.32 (m, 12H), 6.51 (t, J=7.8 Hz, 1H), 3.50 (dd, J=14.4, 17.4Hz, 4H).

N,N′-(p-tolylmethylene)bis(2-phenylacetamide) (8). Yield: 70%. ¹H NMR(400 MHz, DMSO-d₆) δ 8.71 (d, J=8.0 Hz, 2H), 7.13-7.32 (m, 14H), 6.52(t, J=8.0 Hz, 1 Hz, 1H), 3.51 (dd, J=14.4, 15.6 Hz, 4H), 2.29 (S, 3H).

N,N′-((4-methoxyphenyl)methylene)bis(2-phenylacetamide) (10). Yield:62%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (d, J=7.6 Hz, 2H), 7.21-7.31 (m,12H), 6.89 (d, J=8.8 Hz, 2H), 6.51 (t, J=8.0 Hz, 1H), 3.74 (s, 3H), 3.50(dd, J=14.0, 17.2 Hz, 4H).

N,N′-((2-(trifluoromethyl)phenyl)methylene)bis(2-phenylacetamide) (13).Yield: 70%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.87 (d, J=7.2 Hz, 2H), 7.78 (d,J=7.6 Hz, 1H), 7.68-7.73 (m, 2H), 7.54 (t, J=7.6 Hz, 1H), 7.19-7.30 (m,10H), 6.83 (t, J=6.8 Hz, 1H), 3.46 (dd, J=14.0, 17.6 Hz, 4H).

N,N′-((4-(trifluoromethyl)phenyl)methylene)bis(2-phenylacetamide) (14).Yield: 75%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (d, J=7.6 Hz, 2H), 7.72 (d,J=7.6 Hz, 2H), 7.51 (d, J=7.6 Hz, 2H), 7.25-7.30 (m, 10H), 6.58 (t,J=7.2 Hz, 1H), 3.53 (s, 4H).

N,N′-((4-nitrophenyl)methylene)bis(2-phenylacetamide) (15). Yield: 84%.¹H NMR (400 MHz, DMSO-d₆) δ 8.94 (d, J=7.8 Hz, 2H), 8.14 (d, J=8.4 Hz,2H), 7.50 (d, J=8.4 Hz, 2H), 7.16-7.25 (m, 10H), 6.52 (t, J=7.8 Hz, 1H),3.47 (s, 4H).

N,N′-((4-(diethylamino)phenyl)methylene)bis(2-phenylacetamide) (17).Yield: 14%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.60 (d, J=8.0 Hz, 2H),7.21-7.31 (m, 10H), 7.07 (d, J=8.4 Hz, 2H), 6.60 (d, J=8.8 Hz, 2H), 6.43(t, J=8.0 Hz, 1H), 3.44-3.52 (m, 4H), 3.29-3.34 (m, 4H), 1.06 (t, J=7.6Hz, 6H). LC-MS (ESI): m/z 430.3 (M+H)⁺.

N,N′-((4-(dibutylamino)phenyl)methylene)bis(2-phenylacetamide) (18).Yield: 15%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.58 (d, J=8.0 Hz, 2H),7.20-7.31 (m, 10H), 7.06 (d, J=8.8 Hz, 2H), 6.57 (d, J=8.8 Hz, 2H), 6.41(t, J=8.0 Hz, 1H), 3.47-3.48 (m, 4H), 3.22-3.26 (m, 4H), 1.43-1.50 (m,4H), 1.26-1.35 (m, 4H), 0.91 (t, J=7.6 Hz, 6H). LC-MS (ESI): m/z 486.2(M+H)⁺.

N,N′-((4-(piperidin-1-yl)phenyl)methylene)bis(2-phenylacetamide) (19).Yield: 45%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.80 (d, J=8.0 Hz, 2H),7.15-7.32 (m, 14H), 6.51 (t, J=8.0 Hz, 1H), 3.37-3.52 (m, 8H), 1.61-1.83(m, 6H). LC-MS (ESI): m/z 442.3 (M+H)⁺.

N,N′-(phenylmethylene)bis(2-(4-chlorophenyl)acetamide) (21). Yield: 52%.¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (d, J=7.8 Hz, 2H), 7.25-7.41 (m, 13H),6.52 (t, J=7.8 Hz, 1H), 3.50 (s, 4H).

N,N′-((2-fluorophenyl)methylene)bis(2-(4-chlorophenyl)acetamide) (22).Yield: 63%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (d, J=7.2 Hz, 2H), 7.45 (t,J=7.2 Hz, 1H), 7.33-7.37 (m, 5H), 7.25-7.26 (m, 4H), 7.18-7.21 (m, 2H),6.73 (t, J=7.2 Hz, 1H), 3.48 (s, 4H).

N,N′-((4-fluorophenyl)methylene)bis(2-(4-chlorophenyl)acetamide) (23).Yield: 67%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.82 (d, J=7.6 Hz, 2H),7.34-7.37 (m, 6H), 7.27 (d, J=8.4 Hz, 4H), 7.19 (t, J=8.8 Hz, 2H), 6.51(t, J=8.0 Hz, 1H), 3.51 (s, 4H).

N,N′-((4-chlorophenyl)methylene)bis(2-(4-chlorophenyl)acetamide) (24).Yield: 40%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (d, J=7.8 Hz, 2H), 7.42 (d,J=8.4 Hz, 2H), 7.31-7.35 (m, 6H), 7.26 (d, J=8.4 Hz, 4H), 6.47 (t, J=7.8Hz, 1H), 3.50 (s, 4H).

N,N′-(p-tolylmethylene)bis(2-(4-chlorophenyl)acetamide) (25). Yield:68%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.76 (d, J=8.0 Hz, 2H), 7.34 (d, J=8.4Hz, 4H), 7.28 (d, J=8.4 Hz, 4H), 7.17 (q, J=8.0 Hz, 4H), 6.50 (t, J=7.6Hz, 1H), 3.51 (s, 4H), 2.29 (s, 3H).

N,N′-((4-methoxyphenyl)methylene)bis(2-(4-chlorophenyl)acetamide) (26).Yield: 61%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.73 (d, J=7.6 Hz, 2H), 7.34 (d,J=8.8 Hz, 4H), 7.26 (d, J=8.4 Hz, 4H), 7.21 (d, J=8.8 Hz, 2H), 6.90 (d,J=8.8 Hz, 2H), 6.46 (t, J=8.0 Hz, 1H), 3.74 (s, 3H), 3.49 (s, 4H).

N,N′-((2-(trifluoromethyl)phenyl)methylene)bis(2-(4-chlorophenyl)acetamide)(27). Yield: 72%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.04 (d, J=6.0 Hz, 2H),7.81 (d, J=7.8 Hz, 1H), 7.72 (d, J=7.8 Hz, 1H), 7.62 (t, J=7.8 Hz, 1H),7.52 (t, J=7.8 Hz, 1H), 7.27-7.30 (m, 8H), 7.06 (t, J=7.2 Hz, 1H), 3.52(s, 4H).

N,N′-((4-(trifluoromethyl)phenyl)methylene)bis(2-(4-chlorophenyl)acetamide)(28). Yield: 65%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.96 (d, J=7.6 Hz, 2H),7.73 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.4 Hz, 4H),7.28 (d, J=8.4 Hz, 4H), 6.57 (t, J=7.6 Hz, 1H), 3.54 (s, 4H).

N,N′-((4-nitrophenyl)methylene)bis(2-(4-chlorophenyl)acetamide) (29).Yield: 80%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.02 (d, J=7.2 Hz, 2H),8.21-8.23 (m, 2H), 7.57 (d, J=7.8 Hz, 2H), 7.33-7.35 (m, 4H), 7.26 (d,J=6.6 Hz, 4H), 6.55 (t, J=7.8 Hz, 1H), 3.53 (S, 4H).

N,N′-(phenylmethylene)bis(2-(4-(trifluoromethyl)phenyl)acetamide) (30).Yield: 73%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.94 (d, J=7.8 Hz, 2H), 7.63 (d,J=7.8 Hz, 4H), 7.48 (d, J=8.4 Hz, 4H), 7.29-7.37 (m, 5H), 6.55 (t, J=7.8Hz, 1H), 3.63 (s, 4H).

N,N′-((2-fluorophenyl)methylene)bis(2-(4-(trifluoromethyl)phenyl)acetamide)(31). Yield: 66%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.99 (d, J=7.2 Hz, 2H),7.63 (d, J=7.8 Hz, 4H), 7.46 (d, J=7.8 Hz, 5H), 7.36-7.40 (m, 1H), 7.21(t, J=7.8 Hz, 2H), 6.74 (t, J=7.8 Hz, 1H), 3.60 (s, 4H).

N,N′-((4-fluorophenyl)methylene)bis(2-(4-(trifluoromethyl)phenyl)acetamide)(32). Yield: 70%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.95 (d, J=8.4 Hz, 2H),7.63 (d, J=8.4 Hz, 4H), 7.47 d, J=8.4 Hz, 4H), 7.37 (dd, J=5.4, 8.4 Hz,2H), 7.19 (t, J=8.4 Hz, 2H), 6.51 (t, J=7.8 Hz, 1H), 3.62 (s, 4H).

N,N′-((4-chlorophenyl)methylene)bis(2-(4-(trifluoromethyl)phenyl)acetamide)(33). Yield: 75%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.96 (d, J=7.8 Hz, 2H),7.63 (d, J=7.2 Hz, 4H), 7.46 (d, J=7.8 Hz, 4H), 7.43 (dd, J=1.8, 8.4 Hz,2H), 7.34-7.35 (m, 2H), 6.50 (t, J=7.2 Hz, 1H), 3.62 (s, 4H).

N,N′-(p-tolylmethylene)bis(2-(4-(trifluoromethyl)phenyl)acetamide) (34).Yield: 71%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.87 (d, J=8.4 Hz, 2H), 7.63 (d,J=7.8 Hz, 4H), 7.47 (d, J=8.4 Hz, 4H), 7.20 (d, J=8.4 Hz, 2H), 7.15 (d,J=7.8 Hz, 2H), 6.49 (t, J=7.8 Hz, 1H), 3.61 (s, 4H), 2.28 (s, 3H).

N,N′-((4-methoxyphenyl)methylene)bis(2-(4-(trifluoromethyl)phenyl)acetamide)(35). Yield: 76%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.85 (d, J=7.8 Hz, 2H),7.63 (d, J=7.8 Hz, 4H), 7.47 (d, J=7.8 Hz, 4H), 7.24 (d, J=9.0 Hz, 2H),6.91 (d, J=8.4 Hz, 2H), 6.48 (t, J=7.8 Hz, 1H), 3.74 (s, 3H), 3.61 (s,4H).

N,N′-(4-(trifluoromethyl)phenyl)methylene)bis(2-(4-(trifluoromethyl)phenyl)acetamide)(36). Yield: 64%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.08 (d, J=7.2 Hz, 2H),7.73 (d, J=7.8 Hz, 2H), 7.63 (d, J=7.8 Hz, 4H), 7.56 (d, J=7.8 Hz, 2H),7.48 (d, J=7.8 Hz, 4H), 6.60 (t, J=7.8 Hz, 1H), 3.35-3.43 (m, 4H).

Synthetic Method 2.

N,N′-(2-phenylethane-1,1-diyl)bis(2-phenylacetamide) (37). To a wellstirred suspension of 2-phenylacetamide (540 mg, 4 mmol) in dry DCM (2mL) was added the 2-phenylacetaldehyde (240 mg, 2 mmol) andtrimethylsilyltrifluoromethane sulonate (22 mg, 0.1 mmol). The mixturewas vigorously stirred for 12 h at room temperature, diluted withtoluene (4 mL), and filtered. The precipitate was washed several timeswith toluene which was recrystallized with methanol and hexane to givethe final product (140 mg, 19%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.45 (d,J=7.6 Hz, 2H), 7.14-7.28 (m, 15H), 5.55 (t, J=7.6 Hz, 1H), 3.39 (s, 4H),2.93 (d, J=7.2 Hz, 2H). LC-MS (ESI): m/z 373.0 (M+H)⁺.

N,N′-(3-phenylpropane-1,1-diyl)bis(2-phenylacetamide) (38). Yield: 52%.¹H NMR (400 MHz, DMSO-d₆) δ 8.45 (d, J=7.6 Hz, 2H), 7.10-7.31 (m, 15H),5.26 (t, J=7.6 Hz, 1H), 3.38-3.46 (m, 4H), 2.47-2.53 (m, 2H), 1.88-1.94(m, 2H). LC-MS (ESI): m/z 387.3 (M+H)⁺.

(E)-N,N′-(3-phenylprop-2-ene-1,1-diyl)bis(2-phenylacetamide) (39).Yield: 18%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (d, J=7.6 Hz, 2H),7.21-7.37 (m, 15H), 6.41-6.45 (m, 1H), 6.27-6.32 (m, 1H), 6.04-6.09 (m,1H), 3.44-3.52 (m, 4H). LC-MS (ESI): m/z 385.1 (M+H)⁺.

N,N′-((4-isopropoxyphenyl)methylene)bis(2-phenylacetamide) (12). Yield:8.1%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.69 (d, J=7.6 Hz, 2H), 7.18-7.13 (m,12H), 6.87 (d, J=6.8 Hz, 2H), 6.48 (t, J=8.0 Hz, 1H), 4.58-4.61 (m, 1H),3.45-3.53 (m, 4H), 1.25 (d, J=6.0 Hz, 6H). LC-MS (ESI): m/z 417.2(M+H)⁺.

N,N′-((4-bromophenyl)methylene)bis(2-phenylacetamide) (7). Yield: 71%.¹H NMR (400 MHz, DMSO-d₆) δ 8.82 (d, J=7.6 Hz, 2H), 7.53-7.56 (m, 2H),7.21-7.33 (m, 12H), 6.48 (t, J=7.6 Hz, 1H), 3.46-3.54 (m, 4H). LC-MS(ESI): m/z 437.0 (M+H)⁺.

N,N′-((4-ethoxyphenyl)methylene)bis(2-phenylacetamide) (11). Yield: 30%.¹H NMR (400 MHz, DMSO-d₆) δ 8.69 (d, J=8.0 Hz, 2H), 7.18-7.31 (m, 10H),6.88 (d, J=6.4 Hz, 2H), 6.48 (t, J=8.0 Hz, 1H), 3.98-4.03 (m, 2H),3.45-3.52 (m, 4H), 1.31 (t, J=6.8 Hz, 3H). LC-MS (ESI): m/z 403.1(M+H)⁺.

N,N′-(pentane-1,1-diyl)bis(2-phenylacetamide) (40). Yield: 63%. ¹H NMR(400 MHz, DMSO-d₆) δ 8.25 (d, J=8.0 Hz, 2H), 7.19-7.30 (m, 8H), 5.30 (t,J=7.6 Hz, 1H), 3.36-3.44 (m, 4H), 1.56-1.62 (m, 2H), 1.14-1.26 (m, 4H),0.81 (t, J=7.2 Hz, 3H). LC-MS (ESI): m/z 339.1 (M+H)⁺.

N,N′-(hexane-1,1-diyl)bis(2-phenylacetamide) (41). Yield: 73%. ¹H NMR(400 MHz, DMSO-d₆) δ 8.25 (d, J=8.0 Hz, 2H), 7.19-7.30 (m, 8H), 5.29 (t,J=7.6 Hz, 1H), 3.39-3.44 (m, 4H), 1.57-1.59 (m, 2H), 1.18-1.23 (m, 6H),0.82 (t, J=6.8 Hz, 3H). LC-MS (ESI): m/z 353.3 (M+H)⁺.

N,N′-((3-fluorophenyl)methylene)bis(2-phenylacetamide) (4). Yield: 27%.¹H NMR (400 MHz, DMSO-d₆) δ 8.83 (d, J=8.0 Hz, 2H), 7.06-7.42 (m, 14H),6.53 (t, J=7.6 Hz, 1H), 3.47-3.55 (m, 4H). LC-MS (ESI): m/z 377.2(M+H)⁺.

N,N′-((4-isopropylphenyl)methylene)bis(2-phenylacetamide) (9). Yield:15%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.73 (d, J=8.0 Hz, 2H), 7.20-7.32 (m,14H), 6.51 (t, J=8.0 Hz, 1H), 3.46-3.54 (m, 4H), 2.85-2.89 (m, 1H), 1.19(d, J=6.8 Hz, 6H). LC-MS (ESI): m/z 401.2 (M+H)⁺.

N,N′-(2-Phenylethane-1,1-diyl)bis(2-phenylacetamide) (44). To a wellstirred suspension of 2-phenylacetamide (540 mg, 4 mmol) in drydichloromethane (2 mL) was added the 2-phenylacetaldehyde (240 mg, 2mmol) and trimethylsilyltrifluoromethanesulfonate (22 mg, 0.1 mmol). Themixture was vigorously stirred for 12 h at room temperature, dilutedwith toluene (4 mL), and filtered. The precipitate was washed severaltimes with toluene which was recrystallized with methanol and hexane togive the final product (560 mg, 76%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.45(d, J=7.6 Hz, 2H), 7.14-7.28 (m, 15H), 5.55 (t, J=7.6 Hz, 1H), 3.39 (s,4H), 2.93 (d, J=7.2 Hz, 2H). HPLC-MS (ESI): m/z 373.2 (M+H)⁺.

N,N′-(3-Phenylpropane-1,1-diyl)bis(2-phenylacetamide) (45). Compound 45was prepared from 2-phenylacetamide and 3-phenylpropanal using theprocedure for compound 44. Yield: 92%. ¹HNMR (400 MHz, DMSO-d₆) δ 8.45(d, J=7.6 Hz, 2H), 7.10-7.31 (m, 15H), 5.26 (t, J=7.6 Hz, 1H), 3.38-3.46(m, 4H), 2.47-2.53 (m, 2H), 1.88-1.94 (m, 2H). LC-MS (ESI): m/z 387.3(M+H)⁺.

(E)-N,N′-(3-Phenylprop-2-ene-1,1-diyl)bis(2-phenylacetamide) (46).Compound 46 was prepared from 2-phenylacetamide and cinnamaldehyde usingthe procedure for compound 44. Yield: 88%. ¹H NMR (400 MHz, DMSO-d₆) δ8.61 (d, J=7.6 Hz, 2H), 7.21-7.37 (m, 15H), 6.41-6.45 (m, 1H), 6.27-6.32(m, 1H), 6.04-6.09 (m, 1H), 3.44-3.52 (m, 4H). LC-MS (ESI): m/z 385.1(M+H)⁺.

N,N′-((4-(Diethylamino)phenyl)methylene)bis(3-phenylpropanamide) (47).Compound 47 was prepared from 3-phenylpropanamide and4-(diethylamino)benzaldehyde according to the procedure for compound 1.Yield: 66%. ¹H NMR (400 MHz, CD₃OD) δ 8.29-8.30 (m, 2H), 7.17-7.29 (m,10H), 6.92 (d, J=8.4 Hz, 2H), 6.56 (d, J=8.4 Hz, 2H), 6.46 (t, J=8.0 Hz,1H), 2.82 (t, J=7.6 Hz, 4H), 2.42-2.47 (m, 4H), 1.07 (t, J=6.8 Hz, 6H).LC-MS (ESI): m/z 458.2 (M+H)⁺. HRMS (ESI) for C₂₉H₃₆N₃O₂(MH⁺): calcd,458.2802. found, 458.2795.

N,N′-((4-(Diethylamino)phenyl)methylene)bis(3-phenylacrylamide) (48).Compound 48 was prepared from cinnamamide and4-(diethylamino)benzaldehyde according to the procedure for compound 1.Yield: 68%. ¹H NMR (400 MHz, CD₃OD) δ 8.68-8.70 (m, 2H), 7.38-7.58 (m,12H), 7.19 (d, J=8.8 Hz, 2H), 6.79 (d, J=16.0 Hz, 2H), 6.66-6.68 (m,3H), 3.29-3.35 (m, 4H), 1.08 (t, J=7.2 Hz, 6H). LC-MS (ESI): m/z 454.2(M+H)⁺. HRMS (ESI) for C₂₉H₃₂N₃O₂ (MH⁺): calcd, 454.2489. found,454.2487.

N,N′-((4-(Diethylamino)phenyl)methylene)dibenzamide (49). Compound 49was prepared from benzamide and 4-(diethylamino)-benzaldehyde accordingto the procedure for compound 1. Yield: 73%. ¹H NMR (400 MHz, CD₃OD) δ9.24 (d, J=7.6 Hz, 1H), 7.88-7.93 (m, 4H), 7.81 (d, J=8.8 Hz, 2H),7.46-7.65 (m, 9H), 7.20 (m, 1H), 3.70-3.81 (m, 4H), 1.17 (t, J=7.2 Hz,6H). LC-MS (ESI): m/z 402.2 (M+H)⁺. HRMS (ESI) for C₂₅H₂₈N₃O₂ (MH⁺):calcd, 402.2176. found, 402.2167.

N,N′-((4-(diethylamino)phenyl)methylene)bis(2-methylpropanamide) (50).Compound 50 was prepared from isobutyramide and4-(diethylamino)benzaldehyde according to the procedure for compound 1.Yield: 80%. 1H NMR (400 MHz, CD₃OD) δ 7.18 (d, J=8.4 Hz, 2H), 6.70-6.73(m, 2H), 6.56 (s, 1H), 3.35-3.50 (m, 2H), 2.47-2.54 (m, 4H), 1.13-1.16(m, 12H). LC-MS (ESI): m/z 334.2 (M+H)⁺. HRMS (ESI) for C₁₉H₃₂N₃O₂(MH⁺): calcd, 334.2489. found 334.2483.

N,N′-((4-(diethylamino)phenyl)methylene)bis(2,2-dimethylpropanamide)(51). Compound 51 was prepared from pivalamide and4-(diethylamino)benzaldehyde using method 1. Yield: 77%. ¹H NMR (400MHz, DMSO) δ 7.70 (d, J=8.8 Hz, 2H), 7.03 (d, J=8.8 Hz, 2H), 6.62 (d,J=8.8 Hz, 2H), 6.52 (t, J=8.4 Hz, 1H), 3.30-3.33 (m, 4H), 1.12 (s, 18H),1.05-1.08 (m, 6H). LC-MS (ESI): m/z 262.2 (M+H)⁺. HRMS (ESI) forC₂₁H₃₆N₃O₂ (MH⁺): calcd, 362.2802. found 362.2795.

N,N′-((4-(diethylamino)phenyl)methylene)dipentanamide (52). Compound 52was prepared from pentanamide and 4-(diethylamino)benzaldehyde usingmethod 1. Yield: 69%. ¹H NMR (400 MHz, CD₃OD) δ 7.18 (d, J=8.8 Hz, 2H),6.71 (d, J=8.8 Hz, 2H), 6.58 (t, J=8.4 Hz, 1H), 3.33-3.41 (m, 4H), 2.25(t, J=7.2 Hz, 4H), 1.58-1.66 (m, 4H), 1.33-1.43 (m, 4H), 1.14 (t, J=7.2Hz, 6H), 0.95 (t, J=2.8 Hz, 6H). LC-MS (ESI): m/z 362.2 (M+H)⁺. HRMS(ESI) for C₂₁H₃₆N₃O₂ (MH⁺): calcd, 362.2802. found 362.2792.

N,N′-((4-(diethylamino)phenyl)methylene)dihexanamide (53). Compound 53was prepared from hexanamide and 4-(diethylamino)benzaldehyde usingmethod 1. Yield: 76%. ¹H NMR (400 MHz, CD₃OD) δ 7.19 (d, J=8.8 Hz, 2H),6.71 (d, J=8.8 Hz, 2H), 6.58 (t, J=8.4 Hz, 1H), 3.35-3.41 (m, 4H),2.19-2.26 (m, 4H), 1.61-1.68 (m, 4H), 1.32-1.35 (m, 8H), 1.14 (t, J=6.8Hz, 6H), 0.94 (t, J=2.8 Hz, 6H). LC-MS (ESI): m/z 390.3 (M+H)⁺. HRMS(ESI) for C₂₃H₄₀N₃O₂ (MH⁺): calcd, 390.3115. found 390.3108.

N,N′-((4-(diethylamino)phenyl)methylene)dioctanamide (54). Compound 54was prepared from octanamide and 4-(diethylamino)benzaldehyde usingmethod 1. Yield: 68%. ¹H NMR (400 MHz, DMSO) δ 8.21 (d, J=8.0 Hz, 2H),7.07 (d, J=8.8 Hz, 2H), 6.61 (d, J=8.8 Hz, 2H), 6.42 (t, J=8.0 Hz, 1H),3.29-3.31 (m, 4H), 2.06-2.14 (m, 4H), 1.47-1.50 (m, 4H), 1.08-1.24 (m,16H), 1.06 (t, J=7.2 Hz, 6H), 0.94 (t, J=7.2 Hz, 6H). LC-MS (ESI): m/z446.3 (M+H)⁺. HRMS (ESI) for C₂₇H₄₈N₃O₂ (MH⁺): calcd, 446.3741. found446.3734.

N,N′-((4-(diethylamino)phenyl)methylene)bis(decanamide) (55). Compound55 was prepared from decanamide and 4-(diethylamino)benzaldehyde usingmethod 1. Yield: 56%. ¹H NMR (400 MHz, DMSO) δ 8.99 (d, J=9.6 Hz, 1H),8.38 (d, J=8.8 Hz, 1H), 6.99 (d, J=8.8 Hz, 2H), 6.58 (d, J=8.4 Hz, 2H),5.84 (d, J=8.4 Hz, 1H), 3.27-3.30 (m, 4H), 2.33 (t, J=7.2 Hz, 4H), 2.14(t, J=7.2 Hz, 4H), 1.50-1.55 (m, 4H), 1.24-1.28 (m, 24H), 1.06 (t, J=7.2Hz, 6H), 0.87 (t, J=7.2 Hz, 6H). LC-MS (ESI): m/z 502.4 (M+H)⁺.

^(a)Reagents and conditions: (i) adamantan-1-amine, methanol, refluxed,10 h; (ii) heptan-1-amine, methanol, refluxed, 12 h; (iii) p-toluidineor 4-chloroaniline, methanol, refluxed, 12 h; (iv) NaBH₄, methanol, r.t,12 h; (v) acyl chloride or sulfonyl chloride, anhydrous DCM, TEA, r.t,12 h.

The synthetic routes to obtain compounds 55-99 are outlined in Scheme 2above. The commercially available 4-(diethylamino)benzaldehyde wasreacted with adamantan-1-amine in methanol to give A, which, whentreated with NaBH₄ gave the secondary amine B. Finally, the couplingreaction between intermediate B and selected acyl chloride or sulfonylchloride yielded the corresponding compounds 56-66. Takingheptan-1-amine, p-toluidine or 4-chloroaniline as the starting material,the synthesis of target compounds 67-99 was accomplished using aprocedure similar to that utilized for preparing compounds 56-66. Thefinal compounds were purified by flash column chromatography.

General Procedure for Synthesis of Secondary Amine Building Blocks.

(3s,5s,7s,E)-N-(4-(Diethylamino)benzylidene)adamantan-1-amine (A).(3s,5s,7s)-adamantan-1-amine hydrochloride (3.75 g, 20 mmol) was addedslowly to a solution of 4-(diethylamino)benzaldehyde and methanol (50mL). The mixture was stirred and refluxed for 12 h. The reaction mixturewas cooled to room temperature and the solvent was removed byevaporation in vacuum to give the crude compound A, which was used tothe next step without further purification.

(3s,5s,7s)-N-(4-(Diethylamino)benzyl)adamantan-1-amine (B). The crudecompound A was dissolved in methanol (50 mL) and NaBH₄ (1.14 g, 30 mmol)was added. The mixture was continued to stir for 12 h at roomtemperature. The reaction solution was poured into water and extractedwith EA. The combined organic layers were washed with water and brine,and then dried over Na₂SO₄. The mixture was filtered and the solvent wasevaporated in vacuum. The residue was purified by flash chromatographyon silica gel to obtain B (5.8 g, 88%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.13(d, J=8.0 Hz, 2H), 6.62 (d, J=8.0 Hz, 2H), 3.65 (bs, 1H), 3.42-3.48 (m,2H), 3.28-3.39 (m, 4H), 2.05-2.07 (m, 3H), 1.58-1.71 (m, 12H), 1.07 (t,J=6.8 Hz, 6H). LC-MS (ESI): m/z 313.2 (M+H)⁺.

4-(((4-Chlorophenyl)amino)methyl)-N,N′-diethylaniline (C). Yield: 78%.¹H NMR (400 MHz, DMSO-d₆) δ 7.03-7.14 (m, 4H), 6.56-6.62 (m, 4H),6.19-6.22 (m, 1H), 4.06-4.07 (m, 2H), 3.27-3.34 (m, 4H), 1.06 (t, J=6.8Hz, 3H). LC-MS (ESI): m/z 289.0 (M+H)⁺.

Synthesis of Compounds 56-99

N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)benzenesulfonamide(56). The intermediate B (328 mg, 1.0 mmol) in dichloromethane (DCM, 5mL) was chilled in ice with the exclusion of moisture and themtriethylamine (122 mg, 1.2 mmol) was added to it. The resulting solutionwas treated dropwise under stirring with benzenesulfonyl chloride (177mg, 1.0 mmol) also dissolved in DCM over 30 min at 0° C. and them leftovernight at room temperature. The reaction solution was poured intowater and extracted with EA. The combined organic layers were washedwith water and brine, and then dried over Na₂SO₄. The mixture wasfiltered and the solvent was evaporated in vacuum. The residue waspurified by flash chromatography on silica gel to obtain 56 (400 mg,85%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.13 (d, J=8.0 Hz, 2H), 6.62 (d, J=8.0Hz, 2H), 3.65 (bs, 1H), 3.42-3.48 (m, 2H), 3.28-3.39 (m, 4H), 2.05-2.07(m, 3H), 1.58-1.71 (m, 12H), 1.07 (t, J=6.8 Hz, 6H). LC-MS (ESI): m/z453.1 (M+H)⁺.

N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-4-fluorobenzenesulfonamide(57) was prepared in a manner analogous to that for compound 56. Yield:87%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.87-7.89 (m, 2H), 7.38-7.43 (m, 2H),7.21 (d, J=8.8 Hz, 2H), 6.64 (d, J=8.8 Hz, 2H), 4.59 (s, 2H), 3.29-3.36(m, 4H), 1.88-1.93 (m, 9H), 1.42-1.51 (m, 6H), 1.07-1.10 (m, 6H). LC-MS(ESI): m/z 471.0 (M+H)⁺.

N-((3s,5s,7s)-Adamantan-1-yl)-4-chloro-N-(4-(diethylamino)benzyl)benzenesulfonamide(58) was prepared in a manner analogous to that for compound 56. Yield:92%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.83 (d, J=8.4 Hz, 2H), 7.64 (d, J=8.8Hz, 2H), 7.21 (d, J=8.8 Hz, 2H), 6.65 (d, J=8.8 Hz, 2H), 4.60 (s, 2H),3.29-3.37 (m, 4H), 1.88-1.93 (m, 9H), 1.42-1.51 (m, 6H), 1.07-1.11 (m,6H). LC-MS (ESI): m/z 487.1 (M+H)⁺.

N-(3r)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-4-methoxybenzenesulfonamide(59) was prepared in a manner analogous to that for compound 56. Yield:89%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.72-7.74 (m, 2H), 7.21 (d, J=8.8 Hz,2H), 7.07-7.10 (m, 2H), 6.64 (d, J=8.8 Hz, 2H), 4.56 (s, 2H), 3.85 (s,3H), 3.29-3.33 (m, 4H), 1.87-1.92 (m, 9H), 1.42-1.50 (m, 6H), 1.07-1.10(m, 6H). LC-MS (ESI): m/z 483.0 (M+H)⁺.

N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-4-methylbenzenesulfonamide(60) was prepared in a manner analogous to that for compound 56. Yield:86%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.70 (d, J=8.4 Hz, 2H), 7.38 (d, J=8.0Hz, 2H), 7.23 (d, J=8.8 Hz, 2H), 6.65 (d, J=8.8 Hz, 2H), 4.59 (s, 2H),3.30-3.42 (m, 4H), 2.40 (s, 3H), 1.87-1.92 (m, 9H), 1.40-1.50 (m, 6H),1.03-1.12 (m, 6H). LC-MS (ESI): m/z 467.2 (M+H)⁺.

N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-3-methylbenzenesulfonamide(61) was prepared in a manner analogous to that for compound 56. Yield:84%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.58-7.62 (m, 2H), 7.42-7.48 (m, 2H),7.23 (d, J=8.4 Hz, 2H), 6.65 (d, J=8.4 Hz, 2H), 4.60 (s, 2H), 3.30-3.34(m, 4H), 2.39 (s, 3H), 1.87-1.92 (m, 9H), 1.41-1.50 (m, 6H), 1.07-1.11(m, 6H). LC-MS (ESI): m/z 467.1 (M+H)⁺.

N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-4-isopropylbenzenesulfonamide(62) was prepared in a manner analogous to that for compound 56. Yield:71%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.72 (d, J=8.0 Hz, 2H), 7.44 (d, J=8.0Hz, 2H), 7.20 (d, J=8.8 Hz, 2H), 6.64 (d, J=8.8 Hz, 2H), 4.57 (s, 2H),3.27-3.29 (m, 4H), 2.97-3.01 (m, 1H), 1.89-1.92 (m, 9H), 1.41-1.51 (m,6H), 1.19-1.24 (m, 6H), 1.08-1.10 (m, 6H). LC-MS (ESI): m/z 495.2(M+H)⁺.

N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)cyclohexanecarboxamide(63) was prepared in a manner analogous to that for compound 56. Yield:89%. ¹H NMR (400 MHz, CDCl₃) δ 7.03 (d, J=8.8 Hz, 2H), 6.68 (d, J=8.8Hz, 2H), 4.49 (s, 2H), 3.35-3.40 (m, 4H), 2.36-2.42 (m, 1H), 2.22 (d,J=2.4 Hz, 6H), 2.03-2.07 (m, 3H), 1.54-1.76 (m, 14H), 1.18-1.21 (m, 8H).LC-MS (ESI): m/z 423.5 (M+H)⁺.

N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)octanamide (64)was prepared in a manner analogous to that for compound 56. Yield: 81%.¹H NMR (400 MHz, CDCl₃) δ 7.05 (d, J=8.4 Hz, 2H), 6.68 (d, J=8.8 Hz,2H), 4.51 (s, 2H), 3.34-3.39 (m, 4H), 2.25-2.33 (m, 8H), 2.05 (s, 3H),1.60-1.70 (m, 8H), 1.17-1.31 (m, 14H), 0.88 (t, J=6.8 Hz, 3H). LC-MS(ESI): m/z 439.4 (M+H)⁺.

N-((3s,5s,7s)-Adamantan-1-yl)-2-(4-chlorophenyl)-N-(4-(diethylamino)benzyl)acetamide(65) was prepared in a manner analogous to that for compound 56. Yield:79%. ¹H NMR (400 MHz, CDCl₃) δ 7.28 (d, J=8.8 Hz, 2H), 7.14 (d, J=8.4Hz, 2H), 7.09 (d, J=8.4 Hz, 2H), 6.71 (d, J=8.8 Hz, 2H), 4.50 (s, 2H),3.62 (s, 2H), 3.36-3.41 (m, 4H), 2.27 (s, 6H), 2.06-2.07 (m, 3H),1.60-1.70 (m, 6H), 1.21 (t, J=7.2 Hz, 6H). LC-MS (ESI): m/z 465.2(M+H)⁺.

N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-2-phenylacetamide(66) was prepared in a manner analogous to that for compound 56. Yield:70%. ¹H NMR (400 MHz, CDCl₃) δ 7.29-7.32 (m, 2H), 7.22-7.26 (m, 3H),7.10 (d, J=8.4 Hz, 2H), 6.71 (d, J=8.8 Hz, 2H), 4.50 (s, 2H), 3.67 (s,2H), 3.36-3.41 (m, 4H), 2.28 (s, 6H), 2.06-2.07 (m, 3H), 1.60-1.70 (m,6H), 1.21 (t, J=7.2 Hz, 6H). LC-MS (ESI): m/z 431.1 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-heptyl-4-methylbenzenesulfonamide (67) wasprepared in a manner analogous to that for compound 56. Yield: 87%. ¹HNMR (400 MHz, CDCl₃) δ 7.74 (d, J=8.4 Hz, 2H), 7.29-7.33 (m, 2H), 7.09(d, J=8.8 Hz, 2H), 6.61 (d, J=8.4 Hz, 2H), 4.23 (s, 2H), 3.33-3.38 (m,4H), 3.06-3.09 (m, 2H), 2.46 (s, 3H), 1.17-1.39 (m, 16H), 1.10-1.15 (m,3H). LC-MS (ESI): m/z 430.7 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-heptyl-3-methylbenzenesulfonamide (68) wasprepared in a manner analogous to that for compound 56. Yield: 89%. ¹HNMR (400 MHz, DMSO-d₆) δ 7.61 (bs, 2H), 7.49-7.50 (m, 2H), 7.06 (d,J=8.4 Hz, 2H), 6.60 (d, J=8.4 Hz, 2H), 4.14 (s, 2H), 3.28-3.33 (m, 4H),2.96-3.00 (m, 2H), 2.40 (s, 3H), 1.14-1.24 (m, 4H), 1.04-1.08 (m, 12H),0.81-0.83 (m, 3H). LC-MS (ESI): m/z 432.0 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-heptyl-4-isopropylbenzenesulfonamide (69)was prepared in a manner analogous to that for compound 56. Yield: 81%.¹H NMR (400 MHz, CDCl₃) δ 7.77 (d, J=8.4 Hz, 2H), 7.37 (d, J=8.0 Hz,2H), 7.07 (d, J=8.4 Hz, 2H), 6.61 (d, J=8.8 Hz, 2H), 4.24-4.27 (m, 2H),3.34-3.38 (m, 4H), 2.97-3.32 (m, 3H), 1.10-1.38 (m, 22H), 0.85-0.89 (m,3H). LC-MS (ESI): m/z 460.2 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-heptyl-4-methoxybenzenesulfonamide (70) wasprepared in a manner analogous to that for compound 56. Yield: 89%. ¹HNMR (400 MHz, CDCl₃) δ 7.77-7.80 (m, 2H), 7.08 (d, J=8.4 Hz, 2H), 6.99(d, J=8.8 Hz, 2H), 6.61 (d, J=8.4 Hz, 2H), 4.22 (s, 2H), 3.90 (s, 3H),3.33-3.38 (m, 4H), 3.05-3.09 (m, 2H), 1.15-1.40 (m, 16H), 0.83-0.89 (m,3H). LC-MS (ESI): m/z 448.2 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-heptyl-4-isopropoxybenzenesulfonamide (71)was prepared in a manner analogous to that for compound 56. Yield: 61%.¹H NMR (400 MHz, DMSO-d₆) δ 7.76 (d, J=8.08 Hz, 2H), 7.13 (d, J=8.8 Hz,2H), 7.06 (d, J=8.4 Hz, 2H), 6.60 (d, J=8.0 Hz, 2H), 4.10 (s, 2H), 3.86(s, 3H), 3.28-3.34 (m, 4H), 2.93-2.96 (m, 2H), 1.03-1.23 (m, 20H),0.79-0.83 (m, 3H). LC-MS (ESI): m/z 475.4 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-4-fluoro-N-heptylbenzenesulfonamide (72) wasprepared in a manner analogous to that for compound 56. Yield: 77%. ¹HNMR (400 MHz, CDCl₃) δ 7.83-7.87 (m, 2H), 7.16-7.21 (m, 2H), 7.05 (d,J=8.4 Hz, 2H), 6.60 (d, J=8.4 Hz, 2H), 4.25 (s, 2H), 3.33-3.38 (m, 4H),3.08-3.13 (m, 2H), 1.02-1.40 (m, 16H), 0.88-0.92 (m, 3H). LC-MS (ESI):m/z 436.0 (M+H)⁺.

4-Chloro-N-(4-(diethylamino)benzyl)-N-heptylbenzenesulfonamide (73) wasprepared in a manner analogous to that for compound 56. Yield: 83%. ¹HNMR (400 MHz, CDCl₃) δ 7.77 (d, J=8.4 Hz, 2H), 7.48 (d, J=8.4 Hz, 2H),7.06 (d, J=8.8 Hz, 2H), 6.60 (d, J=8.4 Hz, 2H), 4.25 (s, 2H), 3.33-3.38(m, 4H), 3.08-3.12 (m, 2H), 1.04-1.42 (m, 16H), 0.88-0.92 (m, 3H). LC-MS(ESI): m/z 450.7 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-heptyl-1-phenylmethanesulfonamide (74) wasprepared in a manner analogous to that for compound 56. Yield: 63%. ¹HNMR (400 MHz, CDCl₃) δ 7.35-7.40 (m, 5H), 7.16 (d, J=8.4 Hz, 2H), 6.65(d, J=8.4 Hz, 2H), 4.05-4.25 (m, 4H), 3.34-3.39 (m, 4H), 2.93-2.96 (m,2H), 1.03-1.41 (m, 16H), 0.88-0.92 (m, 3H). LC-MS (ESI): m/z 431.2(M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-heptylbutane-1-sulfonamide (75) wasprepared in a manner analogous to that for compound 56. Yield: 60%. ¹HNMR (400 MHz, CDCl₃) δ 7.19 (d, J=8.4 Hz, 2H), 6.66 (d, J=8.8 Hz, 2H),4.31 (s, 2H), 3.40-3.40 (m, 4H), 3.14-3.16 (m, 2H), 2.88-2.92 (m, 2H),1.77-1.81 (m, 2H), 0.88-1.57 (m, 24H). LC-MS (ESI): m/z 398.0 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-heptyl-2-phenylacetamide (76) was preparedin a manner analogous to that for compound 56. Yield: 70%. ¹H NMR (400MHz, DMSO-d₆) δ 7.25-7.33 (m, 5H), 7.08 (d, J=8.4 Hz, 1H), 6.94 (d,J=8.8 Hz, 1H), 6.65-6.69 (m, 2H), 4.47-4.51 (m, 2H), 3.81-3.82 (m, 2H),3.31-3.40 (m, 5H), 3.24-3.28 (m, 1H), 1.12-1.29 (m, 16H), 0.88-0.92 (m,3H). LC-MS (ESI): m/z 396.1 (M+H)⁺.

2-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-N-heptylacetamide (77) wasprepared in a manner analogous to that for compound 56. Yield: 65%. ¹HNMR (400 MHz, DMSO-d₆) δ 7.16-7.35 (m, 4H), 7.17 (d, J=8.4 Hz, 1H), 7.08(d, J=6.4 Hz, 1H), 6.66-6.71 (m, 2H), 4.51 (s, 2H), 3.80-3.81 (m, 2H),3.35-3.39 (m, 5H), 3.26-3.28 (m, 1H), 1.14-1.67 (m, 16H), 0.89-0.92 (m,3H). LC-MS (ESI): m/z 428.8 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-4-(dimethylamino)-N-heptylbenzamide (78) wasprepared in a manner analogous to that for compound 56. Yield: 80%. ¹HNMR (400 MHz, CDCl₃) δ 7.37 (d, J=8.4 Hz, 2H), 7.09 (bs, 2H), 6.64-6.66(m, 4H), 4.55 (s, 2H), 3.32-3.37 (m, 6H), 2.98 (s, 6H), 1.57-1.61 (m,2H), 1.14-1.28 (m, 14H), 0.86 (t, J=7.2 Hz, 6H). LC-MS (ESI): m/z 424.4(M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-heptylcyclohexanecarboxamide (79) wasprepared in a manner analogous to that for compound 56. Yield: 91%. ¹HNMR (400 MHz, CDCl₃) δ 6.97-7.06 (m, 4H), 6.59-6.66 (m, 4H), 4.11-4.46(m, 2H), 3.14-3.37 (m, 6H), 2.47-2.51 (m, 1H), 1.51-1.82 (m, 10H),1.01-1.49 (m, 16H), 0.84-0.90 (m, 3H). LC-MS (ESI): m/z 387.1 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-heptyloctanamide (80) was prepared in amanner analogous to that for compound 56. Yield: 95%. ¹H NMR (400 MHz,MeOD) δ 7.02-7.09 (m, 2H), 6.67-6.87 (m, 2H), 4.49 (s, 2H), 3.24-3.41(m, 6H), 2.43 (t, J=7.2 Hz, 2H), 1.13-1.68 (m, 26H), 0.89-0.95 (m, 3H).LC-MS (ESI): m/z 402.9 (M+H)⁺.

N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)benzenesulfonamide (81) wasprepared in a manner analogous to that for compound 56. Yield: 73%. ¹HNMR (400 MHz, DMSO-d₆) δ 7.64-7.73 (m, 5H), 7.32-7.34 (m, 2H), 6.96-7.05(m, 4H), 6.50 (d, J=7.2 Hz, 2H), 4.63 (s, 2H), 3.23-3.26 (m, 4H),1.02-1.04 (m, 6H). LC-MS (ESI): m/z 428.9 (M+H)⁺.

N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-4-fluorobenzenesulfonamide(82) was prepared in a manner analogous to that for compound 56. Yield:69%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.68-7.70 (m, 2H), 7.44-7.49 (m, 2H),7.33 (d, J=8.8 Hz, 2H), 7.06 (d, J=8.4 Hz, 2H), 6.96 (d, J=8.8 Hz, 2H),6.50 (d, J=8.8 Hz, 2H), 4.62 (s, 2H), 3.22-3.28 (m, 4H), 1.02 (t, J=6.8Hz, 6H). LC-MS (ESI): m/z 446.9 (M+H)⁺.

4-Chloro-N-(4-chlorophenyl)-N-(4-(diethylamino)benzyl)benzenesulfonamide(83) was prepared in a manner analogous to that for compound 56. Yield:65%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.70 (d, J=8.8 Hz, 2H), 7.63 (d, J=8.8Hz, 2H), 7.34 (d, J=8.8 Hz, 2H), 7.07 (d, J=8.8 Hz, 2H), 6.96 (d, J=8.8Hz, 2H), 6.50 (d, J=8.8 Hz, 2H), 4.62 (s, 2H), 3.22-3.28 (m, 4H),1.01-1.06 (m, 6H). LC-MS (ESI): m/z 463.0 (M+H)⁺.

N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-4-methoxybenzenesulfonamide(84) was prepared in a manner analogous to that for compound 56. Yield:55%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.56 (d, J=8.8 Hz, 2H), 7.32 (d, J=8.8Hz, 2H), 7.13 (d, J=8.8 Hz, 2H), 7.05 (d, J=8.8 Hz, 2H), 6.96 (d, J=8.8Hz, 2H), 6.50 (d, J=8.8 Hz, 2H), 4.58 (s, 2H), 3.86 (s, 3H), 3.22-3.27(m, 4H), 1.02 (t, J=6.8 Hz, 6H). LC-MS (ESI): m/z 459.1 (M+H)⁺.

N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-4-methylbenzenesulfonamide(85) was prepared in a manner analogous to that for compound 56. Yield:79%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.31-7.51 (m, 6H), 7.03-7.05 (m, 2H),6.96 (d, J=8.8 Hz, 2H), 6.49 (d, J=8.8 Hz, 2H), 4.59 (s, 2H), 3.22-3.27(m, 4H), 2.42 (s, 3H), 1.02 (t, J=6.8 Hz, 6H). LC-MS (ESI): m/z 443.2(M+H)⁺.

N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-3-methylbenzenesulfonamide(86) was prepared in a manner analogous to that for compound 56. Yield:65%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.38-7.53 (m, 4H), 7.32 (d, J=8.4 Hz,2H), 7.04 (d, J=8.8 Hz, 2H), 6.96 (d, J=8.8 Hz, 2H), 6.50 (d, J=8.8 Hz,2H), 4.61 (s, 2H), 3.22-3.28 (m, 4H), 2.40 (s, 3H), 1.02 (t, J=6.8 Hz,6H). LC-MS (ESI): m/z 443.0 (M+H)⁺.

N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-4-isopropylbenzenesulfonamide(87) was prepared in a manner analogous to that for compound 56. Yield:89%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.56 (d, J=8.4 Hz, 2H), 7.49 (d, J=8.0Hz, 2H), 7.32 (d, J=8.8 Hz, 2H), 7.06 (d, J=8.8 Hz, 2H), 6.96 (d, J=8.8Hz, 2H), 6.50 (d, J=8.8 Hz, 2H), 4.61 (s, 2H), 3.23-3.26 (m, 4H),2.98-3.05 (m, 1H), 1.25 (d, J=6.8 Hz, 6H), 1.03 (t, J=6.4 Hz, 6H). LC-MS(ESI): m/z 471.1 (M+H)⁺.

N,2-bis(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)acetamide (88) wasprepared in a manner analogous to that for compound 56. Yield: 79%. ¹HNMR (400 MHz, DMSO-d₆) δ 7.44 (d, J=8.4 Hz, 2H), 7.32 (d, J=8.4 Hz, 2H),7.09-7.14 (m, 4H), 6.91 (d, J=8.8 Hz, 2H), 6.54 (d, J=8.8 Hz, 2H), 4.71(s, 2H), 3.42 (s, 2H), 3.25-3.31 (m, 4H), 1.05 (t, J=6.4 Hz, 6H). LC-MS(ESI): m/z 442.8 (M+H)⁺.

N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)cyclohexanecarboxamide (89)was prepared in a manner analogous to that for compound 56. Yield: 68%.¹H NMR (400 MHz, DMSO-d₆) δ 7.44 (d, J=8.4 Hz, 2H), 7.11 (d, J=8.4 Hz,2H), 6.89 (d, J=8.4 Hz, 2H), 6.55 (d, J=8.4 Hz, 2H), 4.65 (s, 2H),3.26-3.31 (m, 4H), 2.08 (bs, 1H), 1.36-1.63 (m, 7H), 0.93-1.13 (m, 9H).LC-MS (ESI): m/z 399.4 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-(p-tolyl)benzenesulfonamide (90) wasprepared in a manner analogous to that for compound 56. Yield: 80%. ¹HNMR (400 MHz, DMSO-d₆) δ 7.60-7.72 (m, 5H), 7.04 (d, J=8.0 Hz, 2H), 6.96(d, J=8.8 Hz, 2H), 6.87 (d, J=8.4 Hz, 2H), 6.50 (d, J=8.8 Hz, 2H), 4.60(s, 2H), 3.23-3.28 (m, 4H), 2.23 (s, 3H), 1.03 (t, J=7.2 Hz, 6H). LC-MS(ESI): m/z 408.9 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-4-fluoro-N-(p-tolyl)benzenesulfonamide (91)was prepared in a manner analogous to that for compound 56. Yield: 83%.¹H NMR (400 MHz, DMSO-d₆) δ 7.66-7.68 (m, 2H), 7.43-7.47 (m, 2H), 7.06(d, J=8.0 Hz, 2H), 6.96 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.4 Hz, 2H), 6.50(d, J=8.8 Hz, 2H), 4.60 (s, 2H), 3.22-3.30 (m, 4H), 2.23 (s, 3H), 1.02(t, J=7.2 Hz, 6H). LC-MS (ESI): m/z 427.2 (M+H)⁺.

4-Chloro-N-(4-(diethylamino)benzyl)-N-(p-tolyl)benzenesulfonamide (92)was prepared in a manner analogous to that for compound 56. Yield: 85%.¹H NMR (400 MHz, DMSO-d₆) δ 7.67-7.70 (m, 2H), 7.61-7.63 (m, 2H), 7.06(d, J=8.0 Hz, 2H), 6.96 (d, J=8.8 Hz, 2H), 6.90 (d, J=8.4 Hz, 2H), 6.50(d, J=8.8 Hz, 2H), 4.60 (s, 2H), 3.22-3.28 (m, 4H), 2.23 (s, 3H), 1.02(t, J=7.2 Hz, 6H). LC-MS (ESI): m/z 442.8 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-4-methoxy-N-(p-tolyl)benzenesulfonamide (93)was prepared in a manner analogous to that for compound 56. Yield: 91%.¹H NMR (400 MHz, DMSO-d₆) δ 7.53-7.56 (m, 2H), 7.11-7.13 (m, 2H), 7.04(d, J=8.4 Hz, 2H), 6.96 (d, J=8.4 Hz, 2H), 6.88 (d, J=8.4 Hz, 2H), 6.49(d, J=8.8 Hz, 2H), 4.56 (s, 2H), 3.86 (s, 3H), 3.22-3.27 (m, 4H), 2.23(s, 3H), 1.02 (t, J=7.2 Hz, 6H). LC-MS (ESI): m/z 439.1 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-4-methyl-N-(p-tolyl)benzenesulfonamide (94)was prepared in a manner analogous to that for compound 56. Yield: 94%.¹H NMR (400 MHz, DMSO-d₆) δ 7.50 (d, J=8.0 Hz, 2H), 7.40 (d, J=8.4 Hz,2H), 7.04 (d, J=8.0 Hz, 2H), 6.96 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.4 Hz,2H), 6.49 (d, J=8.8 Hz, 2H), 4.57 (s, 2H), 3.22-3.27 (m, 4H), 2.42 (s,3H), 2.22 (s, 3H), 1.02 (t, J=7.2 Hz, 6H). LC-MS (ESI): m/z 423.0(M+H)⁺.

N-(4-(Diethylamino)benzyl)-3-methyl-N-(p-tolyl)benzenesulfonamide (95)was prepared in a manner analogous to that for compound 56. Yield: 72%.¹H NMR (400 MHz, DMSO-d₆) δ 7.37-7.51 (m, 4H), 7.05 (d, J=8.4 Hz, 2H),6.96 (d, J=8.4 Hz, 2H), 6.86-6.88 (m, 2H), 6.50 (d, J=8.8 Hz, 2H), 4.59(s, 2H), 3.22-3.28 (m, 4H), 2.39 (s, 3H), 2.23 (s, 3H), 1.03 (t, J=7.2Hz, 6H). LC-MS (ESI): m/z 423.4 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-4-isopropyl-N-(p-tolyl)benzenesulfonamide(96) was prepared in a manner analogous to that for compound 56. Yield:86%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.54-7.56 (m, 2H), 7.47 (d, J=8.4 Hz,2H), 7.05 (d, J=8.0 Hz, 2H), 6.96 (d, J=8.4 Hz, 2H), 6.89 (d, J=8.4 Hz,2H), 6.49 (d, J=8.8 Hz, 2H), 4.58 (s, 2H), 3.22-3.28 (m, 4H), 2.98-3.05(m, 1H), 2.23 (s, 3H), 1.25 (d, J=7.2 Hz, 6H), 1.02 (t, J=7.2 Hz, 6H).LC-MS (ESI): m/z 450.9 (M+H)⁺.

N-(4-(Diethylamino)benzyl)-2-phenyl-N-(p-tolyl)acetamide (97) wasprepared in a manner analogous to that for compound 56. Yield: 86%. ¹HNMR (400 MHz, DMSO-d₆) δ 7.17-7.27 (m, 5H), 7.05 (d, J=7.2 Hz, 2H), 6.97(d, J=8.0 Hz, 2H), 6.91 (d, J=8.8 Hz, 2H), 6.54 (d, J=8.8 Hz, 2H), 4.69(s, 2H), 3.35-3.40 (m, 2H), 3.25-3.31 (m, 4H), 2.30 (s, 3H), 1.05 (t,J=7.2 Hz, 6H). LC-MS (ESI): m/z 387.5 (M+H)⁺.

2-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-N-(p-tolyl)acetamide (98)was prepared in a manner analogous to that for compound 56. Yield: 86%.¹H NMR (400 MHz, DMSO-d₆) δ 7.31 (d, J=8.4 Hz, 2H), 7.18 (d, J=8.0 Hz,2H), 7.08 (d, J=8.0 Hz, 2H), 6.98 (d, J=8.0 Hz, 2H), 6.91 (d, J=8.4 Hz,2H), 6.54 (d, J=8.8 Hz, 2H), 4.68 (s, 2H), 3.35-3.38 (m, 2H), 3.25-3.32(m, 4H), 2.30 (s, 3H), 1.05 (t, J=7.2 Hz, 6H). LC-MS (ESI): m/z 420.7(M+H)⁺.

N-(4-(Diethylamino)benzyl)-N-(p-tolyl)cyclohexanecarboxamide (99) wasprepared in a manner analogous to that for compound 56. Yield: 84%. ¹HNMR (400 MHz, DMSO-d₆) δ 7.18 (d, J=8.0 Hz, 2H), 6.94 (d, J=8.0 Hz, 2H),6.89 (d, J=8.4 Hz, 2H), 6.54 (d, J=8.4 Hz, 2H), 4.63 (s, 2H), 3.26-3.31(m, 4H), 2.30 (s, 3H), 2.08-2.14 (m, 1H), 1.35-1.62 (m, 8H), 0.86-1.09(m, 8H). LC-MS (ESI): m/z 379.5 (M+H)⁺.

Methods and Uses

Inventive compounds that conform to Formulae I, I′, II, III, III′ and IVare useful for modulating CB2 receptor activity. The endocannabinoidsystem that is formed of the CB1 and CB2 receptors plays an importantphysiological role glaucoma, cancer, stroke, pain, neuronal disorders,osteoporosis, multiple sclerosis, and autoimmune disorders. The presentinvention focuses on the of small molecule therapeutic agents thatselectively target and modulate the activity of the CB2 receptor, aswell as the use of such compounds to treat multiple myeloma andosteoporosis.

Multiple Myeloma (MM)

CB2 signaling plays an important role in B cell maturation andfunctions. The observation that CB2 is consistently overexpressed inmalignant B cells and MM cells when compared to reactive lymphoid tissueor normal purified B lymphocytes, led to the conclusion that agentscapable of selectively targeting CB2 receptor and capable of modulatingits activity may play a role in the treatment of MM.

For example, FIG. 1 illustrates higher expression of cannabinoidreceptor 2 in various multiple myeloma cells grown in culture usingWestern blot or RT-PCR analysis. This figure also shows that CB1 isprimarily expressed in brain tissue and its expression in MM cells isnegligible.

Several compounds according to the present invention were synthesized asdescribed above. Radiometric binding studies using cultured cellsexpressing CB2 and [³⁵S]-GTP γS binding assays were used to demonstratethe CB2 binding ability of compounds in accordance with the inventionand to identify whether the inventive Formulae I, II or III compoundswere agonist, antagonist or inverse agonists of CB2.

Biological Testing Data

1. Cell Culture and Reagents

Human MM cell lines U266, H929, OPM2, RPMI-8226 and its subline RPMI8226/LR5 (resistant to melphalan), MM.1 S and its subline MM.1 R(resistant to dexamethasone) were cultured as described earlier. Thechemoresistant cell lines were cultured in the presence of melphalan ordexamethasone, and resistance phenotype was confirmed by cellproliferation assays. zVAD-fmk was from Calbiochem (San Diego, Calif.,USA). Cannabinoid ligands SR141716 (CB1 inverse agonist), CP55940(CB1/CB2 agonist), Win55212-2 (CB1/CB2 agonist), SR144528 (CB2 inverseagonist) were provided by NIH-NIDA-NDSP program. The radioligand[³H]-CP55940 used for receptor binding assay was obtained fromPerkin-Elmer (Boston, Mass., USA).

2. Biological Assays

Briefly, the bioassay is carried out using the Perkin Elmer 96-well TopCounter. Competition binding assay was used to evaluate the CB receptorbinding affinity (K_(i)) of the screened ligands by displacing[³H]CP-55940. The procedure is as follows.

The CB receptor binding affinity (Ki) of the in-silico screened ligandsis determined via displacement of [³H]CP-55,940. In competition bindingexperiments, the tested compound dilutions are carried out in duplicatein TME buffer (25 mM Tris, 5 mM MgCl₂, 1 mM EDTA) containing 0.1% (w/v)fatty acid free bovine serum albumin (BSA), pH 7.4. Variousconcentrations of the tested compound are added in the same volume to 3nM [³H]CP-55,940. TME buffer and cell membrane preparations expressingCB receptors (5 μg per well) are added to a final volume of 200 μL. Forthe saturation binding experiments, varying concentrations of[³H]CP-55,940 (0.05-1.5 nM) with or without 2 μM of unlabeled ligands(CP-55,940) are incubated with the receptor membrane preparations todetermine K_(d) and nonspecific binding. After the binding suspensionsare incubated at 30° C. for 1 hr, the reaction is terminated by rapidfiltration through microfiltration plates (Unifilter GF/B filterplate,PerkinElmer), followed by 5 washes with ice cold TME buffer containing0.1% BSA on a Packard Filtermate Harvester (PerkinElmer). The plates arethen dried overnight and 30 μl MicroScint 0 scintillation cocktail isadded to each well of the dried filter plates. The bound radioactivityis then counted using a Perkin Elmer 96-well TopCounter. The K_(i) iscalculated by using nonlinear regression analysis (Prism 5; GraphPadSoftware Inc., San Diego, Calif.), with the K_(d) values for[³H]CP-55,940 determined from saturation binding experiments. This assayis used for determination of the binding affinity parameters (K_(i)) ofligand-receptor interactions for the CB receptor.

A tritiated thymidine incorporation assay was carried out to investigatethe effects of CB2 ligands on cell proliferation. U266, RPMI-8226 (3×10₄cells/well), MM.1 S cells (6×10⁴ cells/well), and their resistantsublines were cultured in 96-well culture plates with or without drugsfor 48 hours. DNA synthesis was measured by ³H-thymidine uptake asdescribed previously.

3. Compounds of the Invention Inhibit Proliferation of Human MM Cells

Cell-based cAMP assays were used to identify the mode by which the CB2ligands according to the present invention modulate CB2 receptor.Briefly, cAMP cell-based assays were used to investigate whether a givenFormula I, I′, II, III, III′ or IV compound was an agonist, antagonist,or an inverse agonist of MM cells expressing the CB2 receptor and tomeasure the anti-MM activity of the compounds of the present invention.Known CB2 agonists, antagonists and inverse agonist were used aspositive controls. The data illustrates that compounds of the inventionbind tightly to the CB2 receptors with K₁ values in the nanomolar range.See Table 3.

TABLE 3A Compounds And Radioligand Binding Data For Compounds OfStructure

Compd R₁ R₂ MW cLog P K_(i) (CB₂), nM^(b, c) K_(i) (CB₁), nM^(a, d)SI^(e)  1 H p-(CH₃)₂N—C₆H₄ 401.50 4.04   777 >20,000  >26  2 H Ph 358.433.93  9,930 NT  3 H o-F—C₆H₄ 376.42 4.08 35,330 NT  4 H m-F—C₆H₄ 376.424.08 12,670 NT  5 H p-F—C₆H₄ 376.42 4.08 10,900 NT  6 H p-Cl—C₆H₄ 392.884.54  3,081 NT  7 H p-Br—C₆H₄ 437.33 4.70  2,226 NT  8 H p-CH₃—C₆H₄372.46 4.45   494   109  9 H p-i-C₃H₇—C₆H₄ 400.51 5.18   85 >20,000 >235 10 H p-CH₃O—C₆H₄ 388.46 3.78   783 >20,000  >26 11 Hp-C₂H₅O—C₆H₄ 402.49 4.13  1,500 NT 12 H p-i-C₃H₇O—C₆H₄ 416.51 4.55  313 >20,000  >64 13 H o-CF₃—C₆H₄ 426.43 4.81 11,780 NT 14 H p-CF₃—C₆H₄426.43 4.81   596 >20,000  >34 15 H p-NO₂—C₆H₄ 403.43 3.87 NB NT 16 Hp-H₂N—C₆H₄ 373.45 2.51 12,550 NT 17 H p-(C₂H₅)₂N—C₆H₄ 429.55 4.76   64 >20,000 >313 18 H p-(C₄H₉)₂N—C₆H₄ 485.66 6.69   221 >20,000  >9019 H p-piperidyl-C₆H₄ 441.56 4.89   595 >20,000  >34 20 Hp-(Benzyl)₂N—C₆H₄ 553.69 7.33   203 >20,000  >99 21 Cl Ph 427.32 5.14 NBNT 22 Cl o-F—C₆H₄ 445.31 5.29 10,850 NT 23 Cl p-F—C₆H₄ 445.31 5.29 NB NT24 Cl p-Cl—C₆H₄ 461.77 5.75   154 >20,000 >130 25 Cl p-CH₃—C₆H₄ 441.355.66   462 >20,000  >43 26 Cl p-CH₃O—C₆H₄ 457.35 4.98   310 >20,000  >6527 Cl o-CF₃—C₆H₄ 495.32 6.02   158 >20,000 >127 28 Cl p-CF₃—C₆H₄ 495.326.02   101 >20,000 >198 29 Cl p-NO₂—C₆H₄ 472.32 5.08 NB NT 30 CF₃ Ph494.43 5.69 NB NT 31 CF₃ o-F—C₆H₄ 512.42 5.83 NB NT 32 CF₃ p-F—C₆H₄512.42 5.83 NB NT 33 CF₃ p-Cl—C₆H₄ 528.87 6.29 NB NT 34 CF₃ p-CH₃—C₆H₄508.46 6.20 NB NT 35 CF₃ p-CH₃O—C₆H₄ 524.45 5.53 NB NT 36 CF₃ p-CF₃—C₆H₄562.43 6.57 NB NT 37 H C₆H₅CH₂ 372.46 3.99 NB NT 38 H C₆H₅CH₂CH₂ 386.494.44  9,319 NT 39 H C₆H₅CH═CH 384.47 4.54  5,683 NT 40 H n-C₄H₉ 338.443.75 35,970 NT 41 H n-C₅H₁₁ 352.47 4.19 18,200 NT   42^(f, g)    2.1 NT  43^(f, h) NT    10.6 44 H C₆H₅CH₂ 372.46 3.99 NB NB 45 H C₆H₅CH₂CH₂386.49 4.44  9,319 NB 46 H C₆H₅CH═CH 384.47 4.54  5,683 NB

TABLE 3B Compounds And Radioligand Binding Data For Compounds OfStructure

K_(i) (CB₂), K_(i) (CB₁), Compd Y MW cLog P nM^(b, c) nM^(a, d) SI^(e)47 CH₂CH₂ 457.61 5.64 231 >20,000  >93 48 CH═CH 453.58 5.80167 >20,000 >119 49 bond 401.50 4.80 688 >20,000  >29

TABLE 3C Compounds And Radioligand Binding Data For Compounds OfStructure

K_(i) K_(i) cLog (CB₂), (CB₁), Compd R^(a′) R^(a″) R^(a′′′) MW PnM^(b, c) nM^(a, d) SI^(e) 50 H CH₃ CH₃ 333.46  3.57 2636 NB 51 CH₃ CH₃CH₃ 361.52  4.69 3553 NB 52 H H C₃H₇ 361.52  4.27  182 >20,000 >109 53 HH C₄H₉ 389.57  5.16  25 >20,000 >800 54 H H C₆H₁₃ 445.68 7.9 146 >20,000 >136 55 H H C₈H₁₇ 501.79 10.0   160 >20,000 >125

TABLE 3D Compounds And Radioligand Binding Data For Compounds OfStructure Compd R K_(i) (CB₂), nM^(b, c) K_(i) (CB₁), nM^(a, d) SI^(e)

B H 19950 NT   130 56

  84 11000   130 57

  25  4268   170 58

 173  2033    11 59

 137  7300    53 60

  47 NB   425 61

  19  8224   432 62

 457 NT 63

  35 NB   >571 64

 638 NT 65

  38 NB   >526 66

  60 NB   >333

67

 2745 NT 68

 2303 NT 69

13000 NT 70

 5193 NT 71

NB NT 72

 1101 NT 73

 6740 NT 74

 273 NT 75

 680 NT 76

 1312 NT 77

 696 NT 78

 1280 NT 79

 212 NT 80

NB NT

G H  6741 NT 81

  20  1773    88 82

  73  1126    15 83

  36  6617   183 84

  14 NB >1,428 85

  37  137    3.7 86

   2.8  866   309 87

 222 NT 88

 136 NB   147 89

 164 NB   121

90

   3.4  514   151 91

   5.6  858   153 92

   3.0  412   137 93

   0.5  1297   2594 94

   5.4  437    80 95

   5.8  218    37 96

   4.3  3365   782 97

  72 NB   >277 98

 107  3200    29 99

 222  202    0.9 ^(a, b)Binding affinities of compounds for CB₁ and CB₂receptor were evaluated using [³H]CP-55,940 radioligand competitionbinding assay. ^(c)NB no binding, K_(i) >20,000 nM. ^(d)NT = not tested.^(e)SI: selectivity index for CB₂, calculated as K_(i)(CB₁)/K_(i)(CB₂)ratio. ^(f)The binding affinities of reference compounds were evaluatedin parallel with compounds 1-99 under the same conditions. ^(g)CB₂reference compound SR 144528. ^(h)CB₁ reference compound SR 141716.

The above compounds in Table 3, however, showed very poor to no bindingactivity to the CBI receptor, thus illustrating the selectivity for CB2.FIG. 2A shows the dose dependent inhibition of DNA synthesis in MM cellsby the known CB2 selective inverse agonists (SR144528 and AM630) and aknown CB2 selective agonist (Hu308) while FIG. 2B illustrates dosedependent inhibition of DNA synthesis in MM cells using a representativecompound (1) according to the present invention.

As demonstrated in FIG. 2A, the known inverse agonist had a modestactivity in inhibiting DNA synthesis in MM cells, while the knownagonist Hu308 showed no effect even at a concentration as high as 10 μM.In contrast, compound (1), showed potent inhibition of DNA synthesis inMM cells a dose-dependent manner (IC_(so): 1.25 μM). c-AMP studiesshowed this compound to be an inverse agonist of CB2. See FIG. 2C. Itwas surprising to discover that compound (1) selectively inhibited thegrowth of two human chemoresistant myeloma cell lines MM.1 R, resistantto dexamethasone and RPMI-226/LR5 resistant to melphalan with IC₅₀values in the range of about 0.6 to 1.2 μM. However, no growthinhibitory effect was seen for their respective parent cells MM.1S andRPMI-8226.

Since drug resistance is a prevalent problem in multiple myelomaclinical treatment, the ability of compounds according to this inventionto overcome chemoresistance of MM cells against conventional drugs suchas dexamethasone or melphalan provides an unexpected advantageousbenefit in MM treatment. The present inventors have hypothesized thatthe cell inhibitory activity of the inventive compounds is due to theirability to activate apoptotic processes. Biological studies by theinventors have shown that while mitogen-activated protein kinase (MAPK)family members activated in response to cell stress are crucial fortriggering apoptosis, are not up regulated in MM cells treated withcompound (1).

Further studies revealed that compound (1) did not have any effect onH2A.X phosphorylation that is critical for DNA damage and apoptosisthrough sustained activation of JNK. However, endoplasmic reticulum (ER)stress-induced transcription factor, CHOP, was transiently upregulated,which promoted upregulation of CHOP-targeted gene death receptor (DR)5a, but not death receptor-4 (DR4). Compound (1) also altered certainproteins that are known to play a role in the cell-growth cycle. Forinstance, contacting cells with various concentrations of compound (1),showed that while number of cells in the G₀-G₁ phase does not change,the number of cells in the S-phase and those in the G2-M phase decreasedin a dose dependent manner. To further explore this observation, thepresent inventors investigated the effect of compound (1) on severalproteins involved in cell G2-M phase transition. In vitro studiesindicate that compound (1) prevents the tyrosine phosphatase Cdc25C fromactivating cyclin-B bound Cdc2 that is responsible for triggeringmitosis and G1 to S phase and G2 to M phase cell transitions.

Taken together, the biological data indicates that compound (1)negatively regulates myeloma cell cycle transitions by modulating theexpression and/or activity of various proteins that are known to beimportant for cell cycle phase transitions, which ultimately result incell growth inhibition and cell death. The role of CB2 in mediating theinhibition of MM cell growth was studied using compound (1) as anexemplary compound according to the present invention. As illustrated byWestern blot analysis (FIGS. 3A and 3B), a stable knockout of CB2,largely abrogated the anti-MM activity of compound (1), suggesting adirect role for the cannabinoid receptor 2 in MM cell death.Pretreatment of MMcells with known agonists Win55212-2 or CP55940 andsubsequent contact of these cells with compound (1) showed thatpretreatment with a CB2 specific agonist attenuated compound (1)mediated inhibition of MM cell growth demonstrating that the CB2receptor is a mediator of MM scell death. See FIGS. 3C and 3D. Theinventors also compared the CB2 receptor modulatory activity of severalknown CB2 ligands to PAM (compound (1)). See, e.g., FIG. 2E.Surprisingly, the present inventors discovered that PAM is an inverseCB2 receptor agonist.

The above results and data illustrated in the figures illustrate thatcompounds that conform to Formulae I, I′, II, III, III′ and IV are anovel class of candidate therapeutics for treating multiple myeloma.

Osteoporosis

In a further embodiment of the invention, Formulae I, I′, II, III, III′and IV compounds are candidate therapeutics for treating a patient orsubject suffering from osteoporosis and in need of treatment. Thepresent inventors surprisingly found that compounds according to thepresent invention suppress osteoclast activity while enhancing theactivity of osteoblasts. The inventive compounds, therefore, provide atwo pronged therapeutic approach to the treatment of osteoporosis, whichdiffers from conventional therapeutic regimens that rely on inhibitionof osteoclast activity to treat osteoporosis.

Using in vitro cell based binding assays, c-AMP regulation and/or³⁵S-GTP-γS binding assays described above, the present inventors testedthe ability of compound (1) to inhibit osteoclast activity and preventbone loss either alone or in combination with the clinically approveddrug Zoledronic in mouse OVX osteoporosis models. Recent data indicatethat compound (1) selectively perturbs CB2 receptor and has exhibitsanti-osteoclast activity on both primary mouse and human bone marrow(BM) preosteoclasts. Compound (1) was also found to be non-toxic whenadministered at concentrations as high as 1 μM.

FIG. 4 illustrates the anti-osteoclast activity of compound (1).Briefly, this figure compares the inhibitory effects of compound (1) anda known CB2 antagonist (XIE-35), from the inventors laboratory onRANKL-induced osteoclast formation in bone marrow mononuclear cells. Itis clear that XIE-35 exerts a weak inhibitory effect on RANKL/M-CSFinduced osteoclast formation as shown by the presence of numerousosteoclasts (arrows). In contrast, compound (1) exerts stronganti-osteoclast effect completely blocking osteoclast formationsuggesting a more favorable spatial orientation that enhances thiscompounds interactions with appropriate amino acid residues in the CB2receptors ligand binding pocket. As stated above CB2 is highly expressedon osteoclasts and is believed to play a role in osteoclast formation,maturation, and modulate processes that are involved in bone resorption.

As a first step in evaluating the therapeutic potential of compounds ofthe present invention as candidate therapeutics for treatingosteoporosis, the present inventors will conduct studies directed tomeasuring the ability of these CB2 ligands inhibit osteoclast (OCL)formation and prevent bone resorption as further explained below.

a. Mouse OCL Formation and Bone Resorption Using Marrow Cells andPreosteoclast Cell Line RAW264.7

Briefly, bone marrow (BM) cells will be flushed from the long bones of4-month-old mice, plated onto petri dishes, and incubated for 48 h inthe presence of M-CSF (100 ng/ml). Nonadherent erythrocytes will beremoved, and the adherent cells will be washed with PBS and resuspendedin culture medium. The resulting M-CSF-dependent bone marrow macrophagescells will be plated onto 96-well plates (5×10⁴ cells per well) in 100μl α-MEM containing 10% FBS, antibiotics, rhM-CSF (10 ng/ml), andrmRANKL (15 ng/ml), with or without a Formulae I, II or III compound andinhibition of osteoclast formation by compounds of the invention will bedetermined fixing the cells and staining the fixed cells with a TRAPstaining kit (Sigma-Aldrich) according to the manufacturer'sinstructions.

The positively stained cells that contain three or more nuclei will becounted as osteoclasts. Mouse OCL formation using RAW264.7 cells(4×10³/well) will be conducted using the similar procedures. To performbone resorption pit assay, murine M-CSF-dependent marrow cells(2×10⁵/well) will be seeded on the dentin slices in 96-well plate andtreated as above for three weeks. The presence of osteoclasts on dentinslices will be confirmed by TRAP staining and bone resorption lacunaewill be stained with hematoxylin.

b. Human Osteoclast Maturation and Function (Bone Resorption Assay)

This assay will measure the effects of our CB2 ligands on human OCLformation and activity. Briefly, nonadherent BM mononuclear cells (10⁵cells/well) will be seeded in 96-well plates in α-MEM containing 20%horse serum, 10 ng/ml rhM-CSF, and 50 ng/ml rhRANKL, in the presence orabsence of CB2 ligands. Half-media changes will be carried out twice aweek where appropriate. Differentiation into OCLs will be assessed bystaining with monoclonal antibody 23c6 that recognizes CD51/61 dimerconstituting the OCL vitronectin receptor, using a Vectastatin-ABC-APkit.

OCL number per well, nuclei per OCL, and OCL size will be counted andmeasured using an inverted microscope and SPOT software. For boneresorption, non-adherent marrow cells (10⁵/well) will be seeded on thedentin slices in 96-well plate and treated as above for four weeks. Boneresorption lacunae will be stained with hematoxylin. All these testswill be conducted using previously published protocols from theinventors [28].

c. Exploration of the Cellular Pathways and Molecular Mechanisms bywhich the CB2 Ligands Exhibit Inhibitory Effects on OCL.

To investigate the molecular mechanism for the anti-osteoclast activityof compounds encompassed by the present invention, granulocytemacrophage colony forming units (CFU-GM) cells that are known to becommitted to osteoclast (OCL) precursor cell will be plated at a celldensity of 5×10⁴/well in 96-well culture plates in the presence of a-MEMmedium containing 10% FCS and 100 pg/ml GM-CSF.

Cells will be incubated with an exemplary Formulae I, II or III compoundat one or more dose for 48 h. Control wells containing cells but no testcompound will also be maintained under identical tissue cultureconditions. Cells will be pulsed with [³H]-TdR 37 kBq/well during thelast 8 h of culture, harvested onto glass-fiber filter mats using anautomatic cell harvester, and counted using a beta plate scintillationcounter. Thymidine uptake will be measured as described in theliterature and the data will be analyzed and presented graphically ascounts per minute (CPM).

Furthermore, given that compounds that conform to Formulae I, II or IIIare selectivity and tight-binding ligands of CB2 receptor, thesecompounds are valuable chemical probes for elucidating the signalpathways that are important in the onset and progression ofosteoporosis. Recent studies have demonstrated the high expression ofcannabinoid receptor CB2 on osteoclasts. In view of the immune monocyteorigin of osteoclasts, targeting CB2 receptor has been proposed toregulate the ratio of activated osteoclast to osteoblast, which iscritical for the seizure of bone loss and pathological fractures inosteoporosis induced by aging or cancers. See for example, Ofek O, etal. Proc Natl Acad Sci USA. 2006; 103:696-701; Idris A I, et al. DrugNews Perspect. 2008; 21:533-40; and Bab I A. Ann N Y Acad. Sci. 2007;116:414-22.

The effect of Formulae I, I′, II, III, III′ and IV compounds ondifferentiation and activity of osteoblasts will also be investigated.Three kinds of experiments: alkaline phosphatase (ALP) activity (anosteoblast differentiation marker), expression of osteoblast relatedgenes (Bsp, Ocn and Runx2) and transactivation of osteoblast-specificOg2 (mouse osteocalcin gene 2), and mineralization assay will be usedfor this study.

To identify specific genes associated with osteoblast developmentMC3T3-E1 preosteoblastic cells MC-4 will be cultured in α-MEM containingascorbic acid (50 μg/ml) for 6 days. Cells will be incubated with anexemplary Formulae I, II or III compound at one or more dose for 48 h.Control wells containing cells but no test compound will also bemaintained under identical tissue culture conditions. Total RNA will beisolated and analyzed by quantitative real-time (RT)-PCR using specificprimers for Bsp (bone sialoprotein), Ocn (osteocalcin), and Runx2 mRNAs,which are normalized to Gapdh mRNA.

In a separate experiment, MC-42 cells that are stably transfected 1.3-kbOg2 (mouse osteocalcin gene 2) promoter driving expression of a fireflyluciferase gene, will be plated in 35-mm plates and cultured inα-MEM-containing ascorbic acid (50 μg/mL) for 15 days. Cells will beincubated with an exemplary compound of the invention at one or moredose for 24 hours. Following incubation, cells are harvested and ssayedfor luciferase and ALP activity. The results were normalized to totalprotein content of the sample.

d. Mineralization Assay

To determine the effect of exemplary Formulae I, I′, II, III, III′ andIV compounds on the mineralization potential of cells in culture, MC-42cells will grown in culture as described above for 15 days. Inorganicphosphate will be added to a final concentration of 5.0 mM in thepresence or absence of a fixed concentrations of a Formulae I, I′, II,III, III′ and IV compound for 48 hours. Following incubation, cells willbe stained using the von Kossa method and imaged by direct scanning ofthe mineralization dish using the ScanMaker 9800 XL as previouslydescribed.

e. In Vivo Studies

Based on the results from ex-vivo studies, compounds identified ashaving potent CB2 modulatory effects will be evaluated in the OVX mousemodel using 25 μg zoledronic acid (ZOL, s.c., Novartis) as a positivecontrol. Briefly, ovariectomized (OVX) C57-BL6 mice (8-wk-old female,Charles River Lab., Wilmington, Mass.) will be divided into 5 groups,(i) sham group; (ii) OVX+vehicle; (iii) OVX+ZOL 25 μg; (iv) OVX+CB2ligand (low dose, ip); and (v) OVX+CB2 ligand (high dose, ip for eachtest compound), with ten (10) mice per group. Dosing with exemplaryFormulae I, I′, II, III, III′ and IV compounds will be commenced on daysix (6) after ovariectomy or sham ovariectomy and dosing will becontinued for four weeks. At the end of the four week study, tibial bonemineral density will be measured for each mouse in the test and shamgroups by microcomputed tomography (micro-CT). Additionally, theosteoclast forming capacity will be evaluated using bone marrow fromtreated and untreated mice. Results from the in vivo study will be usedto advance certain potent compounds to more advanced clinical trials.

f. Osteoclast Formation Bioactivity

A number of compounds according to the invention were selected asrepresentative candidates to be evaluated against RANKL-inducedosteoclast differentiation on RAW 264.7 cells. RAW 264.7 is a mousemonocytic cell line that is used as a standard osteoclastdifferentiation model. As shown in FIG. 5, the inventors tested theeffect of these candidates on osteoclast (OCL) formation using RAW 264.7cells. Each ligand that was tested induced a concentration-dependentinhibition of osteoclastogenesis and all of the tested compoundsgenerally showed strong potency in suppressing OCL formation at 10 μM,with inhibition rates of >95%.

Compounds 84 and 93 also showed good inhibition activity at low dose of1 μM (FIG. 5). Meanwhile, these results indicated that the inhibitionactivities are consistent with the CB₂ binding affinities. Especially,compound 93 showed the strongest inhibition activity, with inhibitionrates of 46%, 97%, and 100% at 0.1, 1, and 10 μM, respectively.

The same techniques demonstrated that compound 17 showed stronginhibition activity, with inhibition rates of 72%, 79%, and 84% at 0.1,1, and 10 μM, respectively.

Cytotoxicity Studies Using Normal Human Cells

The compounds of the invention showed promising inhibition activity withrespect to osteoclastogenesis. To examine whether the impairedosteoclastogenesis in the presence of PAM compounds is due to their celltoxicity, the inventors investigated the cytotoxicity profile of PAMcompounds on normal human cells.

Peripheral blood was drawn in a heparinized syringe from healthy fastingvolunteers who had been without medication for at least 2 weeks. Theperipheral blood mononuclear cell (PBMC) fraction was obtained bygradient centrifugation over Ficoll-Hypaque (Amersham), as described byFeng, R. et al. S. KD5170, a novel mercaptoketone-based histonedeacetylase inhibitor, exerts antimyeloma effects by DNA damage andmitochondrial signaling, Mol. Cancer Ther. 2008, 7, 1494-1505. PBMC werewashed three times with ice-cold PBS, followed by resuspension at5×105/mL in the culture medium supplemented with 10% inactivated FBS, 2mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma).The compounds in a stock solution (50 mMin DMSO) were diluted with theculture medium to application conditions and further used for thetreatment of PBMC for 3 days. The final DMSO concentrations are always0.02%. After treatment for 72 h, cell viability was determined usingtrypan blue exclusion assay. These human cell studies conformed to theguidelines of the Institutional Review Board of the University ofPittsburgh, Pa.

After treatment of these normal cells with compounds 9, 12, 17, 84, and93 for 3 days, the trypan blue exclusion assays indicated that the cellviability was not significantly affected in comparing with the vehiclecontrol group. For instance, compound 17 did not show any cytotoxiceffects at the concentration (1 μM) of 79% inhibition ofosteoclastogenesis, and only slight effects on cell viability wereobserved at high concentration of 10 μM.

Similarly for compounds 84 and 93, cell viability was not significantlyaffected in comparing with the vehicle control group at 1.25 and 2.5 μM,and only some effects on cell viability were observed at highconcentrations of 5 and 10 μM. Compound 84 did not show any cytotoxiceffects at 1.25 μM (97% inhibition rate at 1 μM), and only slight effecton cell viability were observed at high concentration of 5 μM.

These results show that the compounds possess favorable therapeuticindexes and the inhibition of human osteoclastogenesis is not a resultof their cytotoxicity.

Equivalents

While certain embodiments have been illustrated and described, it shouldbe understood that changes and modifications can be made therein inaccordance with ordinary skill in the art without departing from thetechnology in its broader aspects as defined in the following claims.

The present disclosure is not to be limited in terms of the particularembodiments described in this application. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods and compositions within the scope of the disclosure, in additionto those enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this disclosure is not limited toparticular methods, reagents, compounds compositions or biologicalsystems, which can of course vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember.

All publications, patent applications, issued patents, and otherdocuments referred to in this specification are herein incorporated byreference as if each individual publication, patent application, issuedpatent, or other document was specifically and individually indicated tobe incorporated by reference in its entirety. Definitions that arecontained in text incorporated by reference are excluded to the extentthat they contradict definitions in this disclosure.

While several, non-limiting examples have been illustrated anddescribed, it should be understood that changes and modifications can bemade therein in accordance with ordinary skill in the art withoutdeparting from the invention in its broader aspects as defined in thefollowing claims.

We claim:
 1. A method for modulating the activity of a cannabinoidreceptor-2(CB2) in a mammal in need thereof, comprising contacting theCB-2receptor with: (a) a compound of Formula A′

wherein: A is selected from the group consisting of —NR₂, OR,(C₁-C₆)alkyl, and (C₃-C₈) heterocycloalkyl; R is selected from the groupconsisting of (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, and (C₂-C₆) alkenyl; R′ isselected from the group consisting of sub-formula X,(C₃-C₁₄)aryl(C₁-C₆)alkylene-, unsubstituted(C₃-C₁₄)heteroaryl-(C₁-C₆)alkylene-, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,adamantyl and C₆H₄R′″; R″ is

wherein (i) l, m, n, p and q are each an integer 1, (ii) four of G, H,J, L and M are —CH and (iii) the remaining one of G, H, J, L and M isCR_(a)′″, or wherein (i) l, m and q are an integer 1, (ii) n is zero,(iii) p is an integer 2, and (iv) G, H and M are —CH and L is CR_(b)′″;and R_(a)′″ is selected from the group consisting of H, substituted orunsubstituted (C₁-C₆)alkyl, —NH₂, —NH(C₁-C₆)alkyl, —N[(C₁-C₆)alkyl]₂,halogen, (C₁-C₆)haloalkoxy, and (C₁-C₆)alkoxy, wherein when substituted(C₁-C₆)alkyl is present, the (C₁-C₆)alkyl is substituted by one or moresubstituents selected from the group consisting of halogen, hydroxyl,alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy,heterocyclylalkoxy groups, carbonyl, ester, urethane, oxime,hydroxylamine, alkoxyamine, aralkoxyamine, thiol, sulfide, sulfoxide,sulfone, sulfonyl, sulfonamide, amine, N-oxide, hydrazine, hydrazide,hydrazine, azide, amide, urea, amidine, guanidine, enamine, imide,isocyanate, isothiocyanate, cyanate, thiocyanate, imine, nitro group,and nitrile; R_(b)′″ is an unsubstituted aryl; wherein sub-formula X isrepresented by:

wherein Q′ is a bond or a (C₁-C₆)alkyl, G, H, J, L, and M are eachindependently selected from the group consisting of a bond, CR′″—, —N—,—O—, and —S—, and no two adjacent members of G, H, J, L, or M cansimultaneously be —O—, —S—, or N, and

 represents the option of having one or more double bonds, or apharmaceutically acceptable salt thereof; or (b) a compound of Formula B

wherein: R is a (C₁-C₆)alkyl; R′ is selected from the group consistingof (C₁-C₇)alkyl, adamantyl and C₆H₄R′′; R″ is C₆H₄R′″; R′″ is selectedfrom the group consisting of H, (C₁-C₆)alkyl, halogen, and(C₁-C₆)alkoxy, or a pharmaceutically acceptable salt thereof.
 2. Amethod for modulating the activity of a cannabinoid receptor-2 (CB2) ina mammal in need thereof, comprising contacting the CB-2 receptor with acompound, or a pharmaceutically acceptable salt thereof, wherein thecompound is shown in the following table:

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44


3. The method according to claim 2, wherein the compound is selectedfrom the group consisting of Compounds 26-44, or a pharmaceuticallyacceptable salt thereof.
 4. The method according to claim 2, wherein thecompound is selected from the group consisting of Compounds 14, 32 and41, or a pharmaceutically acceptable salt thereof.
 5. The methodaccording to claim 3, wherein the compound is Compound 41, or apharmaceutically acceptable salt thereof.
 6. The method according toclaim 1, wherein the mammal is a human.
 7. The method according to claim2, wherein the mammal is a human.
 8. The method according to claim 1,wherein the compound is a compound of Formula A.
 9. The method accordingto claim 1, wherein the compound is a compound of Formula B.
 10. Themethod of claim 8, wherein A is —NR₂ and R is a (C₁-C₆)alkyl.
 11. Themethod of claim 8, wherein R″ is

wherein (i) l, m, n, p and q are each an integer 1, (ii) four of G, H,J. L and M are —CH, and (iii) the remaining one of G, H, J, L and M isCR_(a)′″, and R_(a)′″ is selected from the group consisting of H,halogen, (C₁-C₆)haloalkoxy, and (C₁-C₆)alkoxy.
 12. The method of claim2, wherein the mammal suffers from multiple myeloma.
 13. The method ofclaim 2, wherein the mammal suffers from osteoporosis.
 14. The method ofclaim 8, wherein the mammal suffers from multiple myeloma.
 15. Themethod of claim 8, wherein the mammal suffers from osteoporosis.
 16. Themethod of claim 9, wherein the mammal suffers from multiple myeloma. 17.The method of claim 9, wherein the mammal suffers from osteoporosis.